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The primary structure of the 2265 residues of bovine plasma fibronectin has been completed. The new sequences reported in this paper are residues 600-868 (269 residues), 1138-1217 (80 residues), 1518 -1599 (82 residues) and 1868-2061 (194 residues). These sequences constitute six type II homology units and two non-monologous connecting strands. Thus, there are fifteen type III homology units in plasma fibronectin. Evidence for two of the three splice variant found in rat liver cels [Schwarzbauer et al. (1983) Cell 35, 421-431] was obtained. No indications of the 'extra' type III domain present in some human fibroblast fibronectins [Kornblihtt et al. (1984) EMBO J. 3, 221-226] was found. Three carbohydrate groups (two glucosamine-based and one galactosamine-based) were identified, giving a total of eight carbohydrate groups in the lowest splice variant of bovine plasma fibronectin. A second free sulfhydryl group (cysteine) was identified. This cysteine, like the first, is in a type III homology unit. HPLC patterns of peptides obtained from the C-terminal 6-kDa fragment suggested that the interchain bridge pattern of fibronectin is antiparallel. The bovine plasma fibronectin sequence is highly homologous to the human cellular fibronectin sequence deduced from the cDNA sequence [Kornblihtt et al. (1985) EMBO J. 4, 1755-1759]. [ABSTRACT FROM AUTHOR]

The reaction of human hemoglobin with carbon monoxide has been investigated near the equilibrium isosbestic wavelength (i.e. 426 nm). As previously reported by others [Gray, R. D. & Gibson, Q. H. (1971) J. Biol. Chem. 246, 5176-5178], in the presence of 0.1 M phosphate pH 7.0 a rise-and-fall kinetic pattern can be observed at this wavelength, which indicates the presence of at least one spectroscopically detectable intermediate species. In this paper we demonstrate that (a) the intermediate species is thermodynamically stable; (b) both phases refer to bimolecular processes; (c) only the initial fast phase is observed when deoxyhemoglobin is reacted with substoichiometric amounts of CO (i. e. final [CO]/[heme] ≤ 0.5); (d) only the second slow phase is observed when hemoglobin that is partially saturated with CO (Y ≤ 0.5) is reacted with saturating CO concentrations; (e) the CO dissociation rate constant measured on the intermediate formed after a partial CO saturation at a final Y ≈ 0.4 has a value similar to that observed starting from the fully liganded form. These results can be accounted for by a two-state allosteric model [Monod, J., Wyman, J. & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118] under the assumption that either (a) 426 nm is an isosbestic wavelength for the T0-R spectral changes but not for the T0-T liganded reaction; or (b) a functional heterogeneity of the two types of subunits is present in the T state and at this wavelength this feature is spectroscopically detectable. [ABSTRACT FROM AUTHOR]

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/ 50 ± 10 mol NF-L and an apparent dissociation constant Kd = 4.3 × 10-7 M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 × 10-6 M with I mol fodrin bound/80 ± 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 × 10-7 M and the maximum binding was I mol fodrin/55 ± 10 mol GFP. [ABSTRACT FROM AUTHOR]

Adult bovine aortic endothelial (ABAE) cells, exposed to serum-free medium, specifically bind 125I-labeled human high-density lipoprotein (125I-HDL). Addition of human lipoprotein-deficient serum (LPDS) reduces the specific binding of 125I-HDL in a concentration-dependent manner, such that LPDS at a concentration of 6 mg protein/ml almost completely inhibits the specific binding of 125I-HDL. ABAE cultures exposed to 125I-labeled LPDS (125I-LPDS) specifically bind two peptides, which appear as minor iodinated components in 125I-LPDS. The binding of these two components is abolished in the presence of excess amounts of unlabeled LPDS or HDL. Preincubation of ABAE cells with 25-hydroxycholesterol (25-HC) results in an increase in the binding of the two 125I-LPDS components, similar to the increase observed in 125I-HDL binding in the presence of 25-HC. These two LPDS components comigrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with apolipoproteins A-I and A-IV of molecular masses 28 kDa and 43 kDa respectively. Furthermore, these two proteins were transfered from the SDS gel to nitrocellulose paper and interacted specifically with anti-(A-I) and anti-(A-IV) sera respectively. When ABAE cultures, pretreated with 25-HC in the presence of LPDS, are subjected to cell-surface iodination, the A-IV appears as one of the major proteins on the cell surface accessible to iodination. The interaction of A-IV with the cell surface of 25-HC-treated cells is not specific to ABAE cells and appears also in human skin fibroblasts. Analysis of the relative amounts of various apolipoproteins in the 125I-HDL bound to ABAE cells demonstrates a decrease in the relative amount of iodinated A-II concomitant with increase in the relative amounts of the other iodinated apolipoproteins, when compared to the composition of the native 125I-HDL. These changes are similar whether the binding is done in the presence or absence of LPDS. It indicates that the decrease in 125I-HDL binding in the presence of LPDS is not due to displacement of the iodinated apolipoproteins A-I and A-IV in the 125I-HDL by unlabeled A-I and A-IV present in LPDS. The results indicate that free apolipoproteins A-I and A-IV, present in LPDS, can displace HDL on the cell surface of ABAE cells. Thus, free A-I and A-IV, present in plasma, control the binding of HDL to endothelial cells and may regulate the process of cholesterol removal from the cells performed by HDL. [ABSTRACT FROM AUTHOR]

As demonstrated by a simple procedure based on indirect immunoprecipitation, proteins retained on heparin- Sepharose 4B from postrnitochondrial supernatants of rat liver and Zajdela hepatoma catalyse the translation of rabbit serum albumin rnRNA in the presence of ribosomal subunits from rather, Zajdela hepatoma or rabbit ceticulocytes. The albumin synthesis shows an optimum at 1.5 mM MgCI2 and 25 mM KCl and requires ATP and GTP. It is significantly stimulated by tRNA and proceeds for more than 2 h. suggesting a high rate of reini- tiation. At the optimum ribosomes : rnRNA ratio of 13: 1, the immunoprecipitable radioactivity exceeds 15 - 20-times the blank values. Fluorography of polyacrylamide slabs after electrophoresis of immunoprecipitates revealed the presence of only complete full size serum albumin without any smaller peptides resulting from premature terminations of polypeptide chains, demonstrating faithful translation. In stained gels only, both heavy and light chains of immunoglobulin G were found, indicating that the assay procedure is highly specific and reliable. The fractionated heterologous protein-synthesizing system described in this paper may be generally useful for studies on the synthesis of specific proteins and factors affecting their rates since, unlike comparable translation assays. a precise calculation of the balance of newly synthesized proteins is possible. [ABSTRACT FROM AUTHOR]

Peptide sequences obtained from hen ovotransferrin are compared with the complete amino acid sequence of the protein deduced from a cDNA sequence (Jeltsch and Chambon, preceding paper). Of the 705 positions of the whole protein 605 can be matched by the peptide sequences. Some possible discrepancies between the two methods are pointed out. The two halves of the chain show marked similarities in their sequences with 37 % identical residues. The positions of the 15 disulphide bridges are shown; there are 6 homologous bridges in each half of the molecule and 3 extra bridges which occur only in the C-terminal half. The terminal residues of the half- molecule fragments obtained by limited proteolysis are identified. The two domains are joined by a 9-residue connecting peptide. Sequence variability has been found at 9 positions. The sequence of hen ovotransfernn is compared with the partial sequence available for human transferrin. From this some tentative conclusions about the identities of the metal-binding residues and about the evolution of transferrin are reached. [ABSTRACT FROM AUTHOR]

The present paper reports data for the effect of pH and D-glycerate 2,3-bisphosphate (D-glycerate-2,3-P2) on the oxygen equilibrium of normal and SH(β93)-modified human hemoglobin. At sufficiently high D-glycerate- 2,3-P2 concentrations, both oxy and deoxy forms of HbA are saturated with the organic phosphate at all pH values between 6 and 9. Furthermore the difference in the affinity for D-glycerate-2,3-P2 between deoxy and oxy hemoglobin remains constant with pH, implying that the pK values of the Bohr effect groups in deoxy and oxyhemoglobin are not affected by the presence of D-glycerate-2,3-P2 on the hemoglobin molecule. In the hemoglobins modified in position β93, the difference in affinity between deoxy and oxy hemoglobin for D-glycerate-2.3-P2 decreases with decrease in pH due mainly to a decrease in the affinity of the deoxy form for D-glycerate-2,3-P2. The effect of the chemical modification appears to be primarily a change in pK of a Bohr group in deoxy hemoglobin which is especially pronounced in the presence of phosphates. [ABSTRACT FROM AUTHOR]

Sheep-serum transferrin shows marked polymorphism and more than 20 alleles have been identified although only 4 or 5 of these have a frequency higher than 1%. Each of the alleles has two bands in starch-gel electrophoresis, corresponding to a major and a minor fraction. This paper describes the isolation and partial characterisation of the two fractions from the transferrin of sheep homozygous for Tf B. The purification consisted of: (a) precipitation by ammonium sulphate, (b) chromatography on CM-cellulose and, finally (c) chromatography on DEAE-Sephadex. The purification procedure had no effect on the electrophoretic mobility of the two fractions and they appeared homogeneous by starch-gel and polyamide-gel electrophoresis and by immuno-electrophoresis. The amino-acid compositions of both fractions were very similar and the sequences of the first eight amino-acid residues: Ser-Pro-Glu-Lys-Thr-Val-Arg-Trp-were identical for both bands. These results, and the fact that the two fractions are found in all genetic variants and always have the same relative mobility strongly suggest that the differences do not lie in the polypeptide chain. From the results of hydrolysis by neuraminidase and assay of sialic acid, the major and minor fractions most probably contain two and three sialic acid residues (exclusively N-acetylneuraminic acid) respectively, thus explaining the different electrophoretic mobilities. The sialic-acid content has been calculated on the basis of a molecular weight of 77500 as determined both by low-speed equilibrium ultracentrifugation and by Archibald's method. The following physico-chemical constants have been established: absorption coefficient at 280nm, A1 mg/cm³1 cm = 1.25 ± 0.01 cm² · mg-1; partial specific volume, &vmacr; = 0.734 cm³ · g-1; sedimentation coefficient s20.wo = 5.15 S; refractive-index increment, dn/dc = 0.173 cm³ · g-1. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,χ) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vχ chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of CNBr fragment H4 of the murine immunoglobulin MOPC 173 (IgG2a,χ) has been determined, thus completing the sequence determination of the entire heavy chain. The H4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CH1 domain, the hinge region and the beginning of CH2. Sequence comparisons suggest that the CH1 domain is highly conserved in evolution, and allows the definition of two additional isotypic-specific regions. [ABSTRACT FROM AUTHOR]

This paper reports some physico-chemical and functional properties of two of the isolated hemoglobin components from trout (Salmo irideus) blood (Hb trout I and Hb trout IV). Both hemoglobins in the oxygenated state are tetrameric at pH between 7 and 7.8 and protein concentration above &assymp; 0.1 mg/ml. The tetramer is extremely stable towards dissociation into subunits and the stability constants, estimated by differential gel filtration, are between 10 and 50 times larger than that of human hemoglobin in phosphate buffer. A very small degree of dissociation is also suggested by the very small extent of hybrid formation even in concentrated KI or triethanolamine. The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. Hb trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity (θ); furthermore upon deoxygenation the value of θ at 222 nm becomes more negative by about 50%. On the ether hand Hb trout I shows a different circular dichroism spectrum and in particular the ellipticity at 222 nm is lower than that of Hb trout IV. Moreover the spectrum is insensitive to the presence of heme ligands. Hb trout IV binds human haptoglobin (Hp) as shown by quenching of the fluorescence of the tryptophanyl residues of Hp upon addition of hemoglobin. The apparent stoichiometry of complex formation corresponds to two Hb tetramers per Hp molecule (while for human hemoglobin the stoichiometry corresponds to &assymp; one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O2 and especially CO combination as studied by flash photolysis. The effect of several third components on the O2 equilibrium of both Hb trout I and IV has been investigated. In general the effect of adding ions or other molecules to a solution of lib trout I is very minor. On the other hand Hb trout IV is more susceptible to changes in solvent composition. Of particular importance is the action of ATP, the physiological organic phosphate, which is without effect on Hb trout I, while it has a clear effect on Hb trout IV. The addition of ATP shifts the Root effect, the characteristic property of Hb trout IV, to higher pH values, thereby acting as one of the allosteric effectors. [ABSTRACT FROM AUTHOR]

Phylogenic research on the haptoglobin of different animal species led us to study the structure of rabbit haptoglobin. Rabbit haptoglobin, whose homogeneity was checked according to several criteria, was submitted to hydrolysis by pronase (72 h, 39°, pH 8.2); the hydrolysate was then filtered through Sephadex G-25, and the resulting glycopeptide fraction was separated on Dowex 50-X2 (H+) into two parts, one (A) not retained by the resin, the other one (B) collected after elution with 0.5 M NaCl. Amino-acid and carbohydrate composition, nature of end groups, and timing of sialic acid release, were determined on fraction A; from the amount of aspartic acid, the molecular weight was estimated to be no less than 2860. Fraction B, which amounted to one third of fraction A, could not be analysed on account of the insufficient quantity available. Quantitative paper electrophoresis at pH 3.5 and 6.2 yielded 8 glycopeptides from fraction A and 2 from fraction B. These various glycopeptides showed notable differences in their amino-acid composition, mainly with respect to glutamic acid, proline, glycine and histidine. On the contrary, no significant differences were found in the carbohydrate composition, with the exception of the oses N-acetylneuraminic acid ratio, which varies from 2 to 10. These results suggest 6 oligosaccharide units to be present in rabbit haptoglobin, as compared to 7 in human haptoglobin, bound to peptidic fractions varying in their amino-acid composition in the neighbourhood of the carbohydrate-peptide linkage. Although the precise nature of this bond has not been ascertained, it does not appear to be an O-glycosidic one. [ABSTRACT FROM AUTHOR]

A major inducible form of heine oxygenase (EC 1.14.99.3) was purified from liver microsomes of chicks pre-treated with cadmium chloride. The purification involved solubilization of microsomes with Emulgen 913 and sodium cholate, followed by DEAE-Sephacel, carboxymethyl-cellulose (CM-52) and hydroxyapatite chromatography, and FPLC through Superose 6 and 12 columns operating in series. The final product gave a single band on silver-stained SDS/polyacrylamide gels (Mr = 33000). Optimal conditions for measurement of activity of solubilized heme oxygenase were studied. In a reconstituted system containing purified heme oxygenase. NADPH -cytochrome reductase, biliverdin reductase and NADPH, the Km for free heme was 3.8 ± 0.5 μM; for heme in the presence of bovine serum albumin (5 mol heme/3 mol albumin) the Km was 5.0 ± 0.8 μM ; and the Km for NADPH was 6.1 ± 0.4 μM (all values mean ± SD, n = 3). Oxygen concentration as low as 15 μM, with saturating concentrations of heme and NADPH, did not affect the reaction rate, indicating that the supply of oxygen is not involved in the physiological regulation of activity of the enzyme. The pH optimum of the reaction was 7.4: at 37 C, the apparent Vmax was 580 ± 44 nmol biliverdin · (mg protein)-1 · min-1 and the molecular activity was 19.2 min-1. Biliverdin IXa was the sole biliverdin isomer formed. In the presence of purified biliverdin reductase, biliverdin was converted quantitatively to bilirubin. Addition of catalase to the reconstituted system decreased the breakdown of heine to non-biliverdin products and led to nearly stoichiometric conversion of heine to biliverdin. Activity of the enzyme in the reconstituted system was inhibited by metalloporphyrins in the following order of decreasing potency: tin mesoporphyrin > tin protoporphyrin > zinc protoporphyrin > manganese protoporphyrin > cobalt protoporphyrin. Protoporphyrin (3.3 or 6.6 μM) (and several other porphyrins) and metallic ions (100 μM) alone had little if any inhibitory effect, except for Hg2+ which inhibited by 67% at 10 μM and totally at 15 μM. Following partial cleavage, fragments of the purified enzyme were sequenced. Comparison of sequences to those derived from eDNA sequences for the major inducible rat and human heme oxygenase showed 69% and 76% similarities, respectively. The histidine residue at position 132 of rat heme oxygenase-1 and the residues (Lys128-Arg136) flanking His132 were conserved in all three enzymes, as well as in the corresponding portion of a fourth less highly similar rat enzyme, heme oxygenase-2. It is suggested that this region plays a critical role in heme binding and in catalyzing the heme oxygenase reaction. [ABSTRACT FROM AUTHOR]

The alkali-stable sialoglycopeptides of secretory immunoglobulins A from human milk have been separated from the alkali-labile glycopeptides by gel filtration and from the asialoglycopeptides by ion-exchange chromatography. The structures of live of them have been determined on the basis of the results obtained by methylation analysis, mass spectrometry and 360 MHz ¹H-NM R spectroscopy. For glycopeptide B, the following structure has been found: ... The other glycopeptides can be considered as extensions of this structure. The following extensions to Gal-6' are proposed: NeuAc(α2 - 6) (glycopeptide A), Gal(β 1 - 3) (glycopeptide D) and Fuc(α1 - 6) (glycopeptide E). Furthermore, in glycopeptide C a fucose residue in (α1 - 3) linkage to GlcNAc-5' could be traced. [ABSTRACT FROM AUTHOR]

Adenosine diphosphate ribose (ADP-ribose) coupled directly to bovine serum albumin by the carbodiimide method resulted in a Iow production of antibodies with a preferential affinity for 5'-AMP. To allow condensation of the ADP-ribose residue in a more specific manner, conjugates of N6-carboxymethylated ADP-ribose (cm6ADP-ribose) and serum albumin were synthesized in two ways. First, adenine-N6-carboxymethylated NAD (cm6NAD) was coupled by carbodiimide to serum albumin nearly exclusively through the carboxyl group as judged from a comparison of the epitope density (number of haptens/albumin) of the conjugate with an antigen prepared from unmodified NAD. However, only incomplete enzymic transformation of the NAD hapten to an ADP-ribose hapten could be achieved due presumably to stearic hindrance. Second, cm6ADP-ribose was synthesized from cm6NAD by treatment with NAD glycohydrolase. This novel compound was coupled to serum albumin or to methylated serum albumin with the aid of carbodiimide. Only conjugates obtained with the last-mentioned protein acceptor elicited significant titers of ADP-ribose antibodies besides a similarly high production of 5'-AMP antibodies. With cm6ADP-ribose coupled to non-methylated serum albumin, 5'-AMP antibodies were produced exclusively, which showed an excellent discrimination between 5'-AMP and 3'-AMP, or AMP residues in nucleic acids. Production of 5'-AMP antibodies was presumably caused by hydrolytic breakdown of the ADP-ribose antigen in the recipient animals. [ABSTRACT FROM AUTHOR]

A specific and sensitive radioimmunoassay for ADP-ribose has been developed on the basis of the selective conversion of ADP-ribose to 5'-AMP by alkaline treatment. Antibodies highly specific against 5'-AMP allowed quantification of ADP-ribose converted to 5'-AMP in the range of 1-40 pmol, and in the presence of large quantities of nucleic acids or 3'-AMP. Poly(ADP-ribose) could also be determined when degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. Determination of the chain length of purified polymer was possible by a parallel determination of ADP-ribose residues after glycohydrolase treatment and of 5'-AMP from the nonreducing end obtained by phosphodiesterase catalyzed hydrolysis. The high specificities of the alkaline conversion of ADP-ribose to 5'-AMP and of the radioimmunoassay for 5'-AMP allowed quantification of protein-bound ADP-ribose residues in crude tissue extracts as verified by comparison with chromatographically purified samples. [ABSTRACT FROM AUTHOR]

The covalent structures of beef myoglobin is being investigated through different experimental approaches. The globin was cleaved by cyanogens bromide. The resulting fragments were fractionated and submitted to tryptic and chymotryptic digestions. The complete amino acid sequence of these peptides has been established. From the overlaps I sequence between chymotryptic and tryptic peptides, it is possible to place most of these peptides of 153 residues of beef myoglobin in unique sequences. The remaining residues have been ordered on the basis of the apparent homology in sequence between beef and horse myoglobins. The horse protein differs from that of beef in 18 positions. All of the amino acid replacements, except three, were confined to amino acid interchange caused by the change of one base in the coding triplet leaving 135 of 153 positions in the sequence invariant in the two proteins. The relations of these data to other is discussed. [ABSTRACT FROM AUTHOR]

A new Lepore-type hemoglobin in a person of Negro descent has been identified. In this person a minor hemoglobin (Hb-LeporeBaltimore) was detected. An abnormal δβ-chain was isolated and fingerprinted. This hybrid-type protein presumably arose as a consequence of unequal crossing over between the δ and β genes, as was concluded for Hb-LeporeBoston and Hb-LeporeHollandia. In the new Lepore-type hemoglobin described here the crossover could be placed between residues δ50 and β86. This type of crossover should occur with the highest frequency if crossing over is equally frequent between any nucleotide and if selection favours none of the crossover types. [ABSTRACT FROM AUTHOR]

Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation process, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectroscopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemoglobins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemichrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly transformed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquornet and the hemichrorne. In order to assign the heme region state of the α and βchains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an α(aquomet)- β(bishistidy1-hemichrorne) state. This demonstrates that the αand β chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrorne state [Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A. & Mazzarella, L. (2002) Proc. Nail Acad. Sci. USA 99, 9801-980611, the quaternary structures of these α(aquornet)- β(bishistidyl- hemichrorne) forms are intermediate between the physiological R and T hemoglobin states. Together, these structures provide information on the general features of this intermediate state. [ABSTRACT FROM AUTHOR]

The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves a subset of insulin-like growth factor binding proteins (IGFBP), which inhibit the activities of insulin-like growth factor (IGF). Through this proteolytic activity, PAPP-A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP-A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP-A to the cell surface. Based on site-directed mutagenesis, we here delineate the PAPP-A GAG-binding site in the C-terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP-A with heparin. In a model of PAPP-A CCP3-CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP-A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP-A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP-A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP-A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP-A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP-A. [ABSTRACT FROM AUTHOR]

Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis is induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin was demonstrated to bind to lipopolysaccharide (LPS) from Gram-negative bacteria and to lipoteichoic acid from Gram-positive bacteria. Binding of hemolin to smooth-type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and Rc) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS was partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate. Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O-specific antigen and the outer-core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern-recognition protein with broad specificity in the defense against micro-organisms. [ABSTRACT FROM AUTHOR]

The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5′ flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5′ flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5′ flanking sequence from -74 to -46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5′ flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression. [ABSTRACT FROM AUTHOR]

Reports on the new panel of monoclonal antibodies of Serotec Ltd. Usage of the antibodies; Recognition of cell-surface antigen and capillary endothelial cells by the QBEND/10 antibody; Use of CD34 antibody in immunocytochemical studies of vascular endothelium.

In Drosophila melanogaster, metamorphosis and reproduction are thought to be supported in large by two immunologically distinct hexameric storage proteins (hexamerins), larval serum protein 1 (LSP-1), a mixed hexamer of three closely related subunits, Lsp-l(alpha;, β and γ) and larval serum protein 2 (LSP-2), a homohexamer of Lsp-2 subunits. To understand the structural and functional differences between these two storage hexamers, the nucleotide sequence of the coding region of the Lsp-1β gene was determined for comparison with LSP-2 and a number of other arthropod hexamerins. The G+C content of the coding sequence is 55 %, with 92.8 % of the codons containing G or C in the third position. Conceptual translation of the Lsp-1β open reading frame revealed a 789-amino-acid polypeptide of 94465 Da. The amino acid sequence of Lsp-1β is 65.8 % identical to that of calliphorin, the major hexamerin of the blowfly, Calli phora vicina, and only 35.2% identical to Drosophila Lsp-2. This greater similarity to calliphorin is also reflected in high aromatic amino acid and methionine contents, in contrast to LSP-2 which is enriched to a lesser extent only in aromatic amino acids. Lsp-1β is also more closely related to calliphorin with respect to the protein domain structure, the presence of a single intron in its gene, and the absence of glycosylation sites. However, phylogenetic analysis based on multiple alignments revealed that LSP-1/calliphorin and LSP-2 form a distinct dipteran clade whose members are more similar to each other than to any previously sequenced lepidopteran hexamerin or arthropod hemocyanin. [ABSTRACT FROM AUTHOR]

Beyond their critical role in thrombosis, platelets perform important functions in vascular remodeling, inflammation, and wound repair. Many of these functions are executed by molecules expressed by activated platelets. A novel molecule, activated-platelet protein-1 (APP-1), was identified by a monoclonal antibody against activated rabbit platelets. When platelets were stimulated by thrombin, A23187 or ADP, APP-1 was expressed on the platelet surface. APP-1 was also detected in whole cell lysates of platelets, but not on the external surfaces of resting platelets. With maximal activation by thrombin, 15900 ± 2800 molecules APP-1 were expressed/platelet. A 2.3-kb cDNA fragment containing a partial coding sequence for APP-1 was isolated from a rabbit bone marrow library by expression cloning with the anti-APP-1 monoclonal antibody. When expressed as a recombinant fusion protein in bacteria, .APP-1 bound specifically to poly(A)-Sepharose. The full-length cDNA coding for human APP-l, obtained by DNA hybridization techniques, showed 98.7% amino acid sequence identity with the rabbit protein. Northern analysis with human APP-1 identified a 3.7-kb mRNA transcript in megakaryocytic lines that express transcripts for platelet proteins. Human APP-1 has four ribonucleotide binding domains with ribonucleoprotein 1 and 2 motifs. By virtue of its ribonucleotide binding domains, APP-1 is structurally related to polyadenylate-binding protein, which regulates translation initiation and polyadenylate shortening, and to nucleolysin, a specific effector molecule found in the granules of cytotoxic T lymphocytes. [ABSTRACT FROM AUTHOR]

Binding equilibria for the interactions of the medium-chain fatty acid anions, laurate and myristate, with defatted human serum albumin have been investigated under yawing environmental conditions such as ionic strength and pH. Since these ligands bind strongly to albumin (Kass ≈ 107 M-1), conventional equilibrium dialysis is not a feasible method for these investigations. Consequently, we employed a dialysis method, allowing determination of very low concentrations of unbound ligand by measuring the rate of exchange of labelled ligand across a dialysis membrane under conditions of chemical equilibrium. Over a range of ionic strength, 8–68 mM, the binding of the first few molecules of laurate to albumin was weakened with increasing ionic strength, whereas the binding of subsequent molecules seemed to proceed independently of ionic strength. The binding of myristate by albumin, however, appeared to be independent of ionic strength in the observed range of concentrations. The influence of pH in the range 5.1–9.0 on the binding of the two fatty acid anions by albumin was more complicated. The first molecule of laurate appeared to bind with a slightly weaker affinity to albumin at low pH, compared to pH 7 and high pH, while the trends for the following molecules varied. The binding of myristate (irrespective of concentration) seemed to strengthen monotonously with pH, but this conclusion depends critically on the interpretation of the kinetic behaviour of the myristate anion. We have previously shown [Pedersen, A. O., Honoré, B. & Brodersen, R. (1990) Eur. J. Biochem. 190, 497–502] that the strength of binding of the first few molecules of the two fatty acid anions to albumin decreases with increasing temperature, whereas binding of subsequent molecules seems to proceed independently of temperature. We explain these findings as follows. The binding of the first few (3 or 4) molecules of the C12 laurate anion is clearly driven by formation of ionic bonds between the fatty acid anion and positively charged groups, such as lysine residues, in the albumin molecule, whereas the binding of subsequent molecules of laurate seems to depend more on hydrophobic interactions. In the case of the C14 myristate anion, the binding of the first few (only 1 or 2) molecules may depend on ionic forces, but binding of the following molecules of myristate seems to depend on hydrophobic interactions only. Thus, multiple binding of both ligands is considered to be an association between fatty acid anions stacked on the protein molecule and/ or it may take place in hydrophobic regions of the albumin molecule. This type of binding will contribute entropy rather than enthalpy to the total binding energy. [ABSTRACT FROM AUTHOR]

An anti-venom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action. CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23–25-kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues. The neutralizing effect of purified C1CS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex. The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system. [ABSTRACT FROM AUTHOR]

In an attempt to improve our understanding of the complex interplay between cell compartments and chemical species during cellular uptake of iron from transferrin, we designed a computer simulation program based on current models of receptor-mediated endocytosis and pinocytosis. The program calculates and visualizes, as a function of time, the changes in transferrin, apotransferrin, and iron concentrations occurring in all relevant cellular compartments during cellular iron acquisition from transferrin. Simulation of literature data showed that the program generates results that are in accordance with experimental data. Furthermore, from measurements of the uptake of [carboxyl-14C]dextran we could utilize the program to suggest rate constants characteristic for the pinocytic process in rat reticulocytes. Moreover, simulations indicate that the apparent difference in the iron uptake process observed between reticulocytes arid hepatocytes may be explained by the contribution made by pinocytosis to the iron uptake process. Finally, the present program should have potential as an educational tool during introduction to the field of receptor-mediated endocytosis in general and to cellular iron metabolism in particular. [ABSTRACT FROM AUTHOR]

Pregnancy zone protein (PZP) is quantitatively the most important pregnancy-associated plasma protein and it has strong similarity to a@-macroglobulin. Since a.@-macroglobulin is a binding protein for transforming growth factors-β(TGF-β), it was of interest to test whether the related protein. PZP. also binds to these growth-regulatory proteins. Using affinity-labelling methods, we demonstrate that PZP binds both TGF-β1 and TGF-β2 and that the binding characteristics are similar to those of the TGF-β-α2-macroglobulin interaction. TGF-β2 and TGF-β1 bind to PZP in a predominantly noncovalent manner in vitro. TGF-β1 and TGF-β2 bind to both the dimeric and tetrameric forms of PZP. Our studies also indicate that PZP binds TGF-β2 with higher affinity than TGF-β1. Finally. we demonstrate that PZP inhibits the binding of TGF-β1 and TGF-β2 to their cell surface receptors. The increased level of PZP during pregnancy may affect the action of TGF-β by regulating the distribution, clearance and/or general availability of TGF-β. The preferential binding of TGFβ2 over TGF-β1 by PZP implies that PZP may differentially regulate the action of TGF-β1 and TGF-β2. [ABSTRACT FROM AUTHOR]

Factor-Xa-catalyzed prothrombin activation is greatly accelerated by negatively charged phospholipids plus calcium ions. In 1990, we reported that neutral phosphatidylcholine membranes also stimulated prothrombin activation [Gerads, I., Govers-Riemslag, J. W. P., Tans, G., Zwaal, R. F. A. & Rosing, J. (1990) Biochemistry 29, 7967--7974]. In the present study, we have performed a detailed analysis of the prothrombin-converting activity of phosphatidylcholine membranes. Stimulation of prothrombin activation by phosphatidylcholine vesicles was particularly observed (a) with phosphatidylcholine molecules that contained unsaturated hydrocarbon side chains, (b) in the presence of factor Va, (c) at low ionic strength and (d) when Ca2+ were present in the reaction medium. It is unlikely that the prothrombinase activity of phosphatidylcholine preparations was due to contaminating anionic phospholipids. This is concluded from the fact that thin-layer chromatographic analysis showed that dioleoylphosphatidylcholine [(Ole)2GroPCho] contained less than 0.1 mol/100 mol anionic phospholipid, and that incorporation of such amounts of anionic lipids in (Ole)2-GroPCho membranes hardly increased their prothrombin-converting activity. At low ionic strength and in the presence of factor Va and Ca2+ (Ole)2GroPCho membranes accelerated prothrombin activation about 100-fold. At ionic strength (I) 0.06, prothrombin activation on 100 µM (Ole)2-GroPCho was characterized by a Km for prothrombin of 2 µM, a Vmax of 3020 IIa min-1 · Xa-1 and a Kd for factor XaVa complex formation at the membrane surface of 7.5 nM. Prothrombin activation on (Ole)2GroPCho membranes was drastically reduced when the ionic strength was increased. The inhibition at high ionic strength could be explained by an effect on the Kd for XaVa complex formation which increased from 7.5 nM at I = 0.06 to 100 nM at I = 0.22. Prothrombin activation on (Ole)2GroPCho required Ca2+ and was dependent on the presence of y-carboxyglutamic acid domains in prothrombin and factor Xa. This indicates that similar interactions may account for the assembly of prothrombinase complexes on phosphatidylcholine and on anionic lipid-containing membranes. [ABSTRACT FROM AUTHOR]

The immunotoxin-enhancing properties of monensin and of human-serum-albumin-monensin conjugates are severely impaired in the presence of human serum. In this study we have therefore investigated the interaction between serum proteins and monensin leading to the inactivation of monensin function as ummunotoxin potentiator. We found that the binding of monensin-specific mAb to thioether-cross-linked or disulfide-cross-linked protein-monensin conjugates is negatively affected by serum, as indicated by immunoenzymic (ELISA) and radioimmunobinding analysis. Size-exclusion chromatography of serum samples indicated that the greatest blocking effect is due to protein components of 40–90 kDa eluting as a broad peak (peak 4). Analysis of the proteins contained within peak 4 by ion-exchange chromatography followed by microsequencing revealed that the major components of peak no. 4 were transferring, human serum albumin and immunoglobulin fragments. Investigations on the nature of the interactions between serum proteins and monensin leading to monensin inactivation were conducted by affinity chromatography of serum on immobilized human-serum-albumin-monensin conjugates, size-exclusion chromatography. SDS/PAGE analysis of serum-treated human-serum-albumin-monensin conjugates, and evaluation of the stability of immobilized human-serum-albumin-bound 125I-monensin following treatment with serum. Addition of esterase inhibitors (e.g. EDTA, 4-nitorphenyl phosphate) or prior treatment of the serum at 56°C partially reversed the serum effects observed. We conclude that serum proteins block the immunotoxin-enhacning effect of monensin and of human-serum-albumin-monensin conjugates by multiple mechanisms involving hydrophobic and covalent interactions and enzyme-mediated cleavage of protein-bound monensin. [ABSTRACT FROM AUTHOR]

The blood of the teleost Chelodonichthys kumu, living in the temperate waters of New Zealand, contains a single hemoglobin. The complete amino acid sequence of the α and β chain has been established. The presence of a reactive Cys in the external positionβCD8(49) causes polymerization through intermolecular disulfide bridges between β chains, with no alteration of functional features. C. kumu Root-effect hemoglobin displays very low or no subunit co-operativity in the physiological pH range. Kinetic experiments on the oxygen dissociation and binding of carbon monoxide show a marked, pH-dependent functional heterogeneity of the two chains, which contributes to the observed reduction of co-operativity. In contrast, kinetic heterogeneity was not observed in the process of CO dissociation, indicating that functional differences between the subunits are detectable only for the dynamic ligand association pathway. The allosteric effector, ATP, seems to increase the pK4, of the proton-linked effect on the slow-reacting subunit, affecting the quaternary equilibrium through stabilisation of the T state at lower pH, rather than enhancing the functional heterogeneity itself. In position E11 of both chains, Val (usually present at the distal side of the heme), is substituted by lie. Although this residue has been shown not to significantly alter ligand binding to the α chain, to some extent it can perturb the access of oxygen to the β chain. Thus this substitution may be the main reason for subunit functional heterogeneity. [ABSTRACT FROM AUTHOR]

The focus of this review is on the ankyrin gene family, key elements in the interaction of the spectrin-based membrane skeleton with the plasma membrane in a variety of tissues and multicellular organisms. The structure/function relationships of ankyrin molecules are reviewed, illustrating how these proteins are uniquely suited to serve as adaptors between the membrane skeleton and a number of integral membrane proteins. Advances in the understanding of ankyrin biology in the brain are discussed and used to show how ankyrins may be involved in the establishment and/or maintenance of specialized plasma membrane domains. Finally, recent research in hematological and neurological disorders are reviewed, suggesting that ankyrins have a role in the development of human disease. [ABSTRACT FROM AUTHOR]

Specific antibodies against raplA and raplB small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with afffinity-purified antibodies against raplA or raplB demonstrated the presence of multiple immunoreactive proteins in the 22- 23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong raplB immunoreactivity, whereas raplA protein was present in much lower amounts. In the cytosol of HL60 cells, only small amounts of raplA and rapl B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rapl ARas proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, raplA and raplB proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells. [ABSTRACT FROM AUTHOR]

Studies on the functional and structural properties of the cdc2 kinase, a key cell-cycle regulator, have been possible thanks to the availability of cdc2-specific immunoreagents. In an attempt to elucidate the biochemical regulation of the cdc2 kinase in more detail, we have raised a series of novel mouse monoclonal antibodies against human recombinant cdc2 protein. The five Mab reported here can be subclassified into two groups according to their interspecies cross-reactivity and distinct immunoprecipitation patterns. Thus, the target epitopes of Mab POH-1, POH-2 and POH-7 (group 1) appear to be limited to a few mammalian species and the fraction of cdc2 immunoprecipitable by these Mab from cellular extracts is considerably enhanced by denaturation. In contrast, the POH-3 and POH-8 (group 2) Mab recognize a denaturation-sensitive epitope on cdc2 which is present in all tested mammalian species. More importantly, each of the two groups of Mab immunoprecipitate forms of cdc2 associated with a characteristic set of cellular proteins, none of which appears to be cyclin A or cyclin B. None of the antibodies precipitated a histone-H 1 or casein-kinase activity, although an activity which phosphorylated some of the coprecipitated proteins was coprecipitated with the group 2 Mab. These novel Mab did not interfere with the association of cdc2 with cyclin A in vitro and efficient immunoprecipitation of a panel of cdc2 mutant proteins suggests that the target epitopes may not involve amino acid residues essential for currently known cdc2 functions. The results of the present study provide evidence for the existence of additional forms of the cdc2 protein in exponentiaIly growing human cells, distinct from both the monomeric and the cyclin-bound cdc2 identified so far. [ABSTRACT FROM AUTHOR]

Equilibria of the binding of palmitate to serum albumin in adults are studied by dialysis-exchange-rate determinations. The results are used for a description of binding equilibria of fatty acids in general, as follows. 1. The reserve albumin concentration, p, for binding of palmitate is used as an approximate measure of p*, the reserve albumin concentration for binding of mixed fatty acids present in serum. 2. The total availability of fatty acids is defined as C*/p*, where C* is the total concentration of non-esterified fatty acid. 3. The fatty-acid-binding property of albumin is described by V = p*/P-αC*/P, where P is the albumin concentration. The numerical value of α is -0.05. The above parameters are measured in sera from four healthy volunteers, in whom large variations of serum fatty acid concentration occurred. A group of 64 healthy students showed considerable variation of L* from one individual to another. It is found that L* decreases significantly with increasing body mass index (body mass divided by the square of the body length). In 42 patients with diabetes type I, L* was independent of body mass index. These findings are consistent with a previously formulated hypothesis of the mechanism of obesity. [ABSTRACT FROM AUTHOR]

1. The investigation of human ceruloplasmin by spectral methods (EPR and spectrophotometry) demonstrated that type 2 Cu2+-containing centres occur not in one, but in two stable forms, differing in EPR and optical spectra. The differential optical spectra of these forms were recorded and the differences in molar absorption coefficients determined. 2. By the EPR method, it was shown that both forms of these centres exist in the blood serum of control donors, as well as in the serum of patients. The relative content of these forms depends on the organism physiological state or on the presence of some pathological condition. 3. The ferroxidase activity of ceruloplasmin against hemoglobin was proved spectrophotometrically. The involvement of other serum proteins in this process cannot be ruled out. The conformational state of ceruloplasmin molecules plays an essential role in its oxidase activity. [ABSTRACT FROM AUTHOR]

Null alleles of all three genes (α, β and γ) coding for the major serum protein of Drosophila larvae, larval serum protein 1, have been characterized at the protein level and by analysis of their RNA and DNA. Each allele completely lacks one polypeptide chain. β shows an unaltered gene but reduced levels of RNA, α shows restriction-fragment-length polymorphism and shows low levels of transcript, while γ′ is a deletion. The three null alleles have been combined to give a strain without the gene for larval serum protein 1 which survives. [ABSTRACT FROM AUTHOR]

Two forms of neutral — alkaline high-molecular-mass proteinase (termed A1 and A2) have been purified from human erythrocytes by a procedure including a DEAE-cellulose batchwise treatment of erythrocyte cytosol, gel filtration and DEAE-cellulose chromatography. Bot Enzymes show distinctive properties of multicatalytic proteinases. They have an apparent molecular mass of 700 kDa and are composed by eight major subunits (23-32 kDa). Both enzymes show a proteinase activity toward casein and hydrolyze synthetic peptides with tyrosine, arginine or lysine at the P1 position. Among the synthetic peptides tested, the tetrapeptide succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin and tripeptides with arginine in the P1 position (benzyloxycarbonyl-valyl-leucyl-arginyl-4-methoxy-2-naphthylamide and benzyloxycarbonyl-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamide) are the most effective substrates. The proteinases are devoid of amino and diaminopeptidase activity. Both enzymes are completely inhibited by hemin, chymostatin and thiol-group reagents. However, the enzymes can be distinguished by the isoelectric point, the different effect of nucleotides, glutathione disulphide, sodium dodecyl sulfate and cations on the catalytic activity [ABSTRACT FROM AUTHOR]

The major enamelin protein component present in EDTA or EDTA/guanidine hydrochloride extracts of developing bovine enamel has a molecular mass of about 67 kDa; it has an amino acid composition similar to that of bovine serum albumin and reacts with potyclonal and monoclonal antibodies to albumin. Two-dimensional separation of the components in the enamclin extract by isoelectric focusing and SDS/PAGE reveal that the major ≈ 67-kDa component and almost all of the minor Coomassie-staining protein components of ≈ 67 kDa, as well as many of the other minor components with different molecular masses, also react with polyclonal and monoclonal antialbumin. The ≈ 67-kDa band eluted after SDS/PAGE, as well as the major ≈ 67-kDa spots eluted after two dimensional separation, were found to have N-terminal amino acid sequences identical to that of bovine serum albumin. Albumin accounted for at least 70- 80% of the total protein content of the enamelin extract and was essentially the only protein in the ≈ 67-kDa component. The serum proteins α-2 HS glycoprotein, γ-globulin and fetuin, and the proline-rich salivary protein termed P-B were also identified in the enamelin extract. The serum proteins and the salivary protein account for > 95% of the proteins in the enamelin extracts. Of the remaining very small amounts of non-serum or salivary protein isolated from the enamelin extracts, three minor components were isolated which had N-terminal amino acid sequences which were not similar to any known protein in the protein sequence data base and could therefore conceivably be true 'enamelins' synthesized by ameloblasts. One additional protein had the first five N-terminal amino acids and residue 8 of amelogenin, residues 6 and 7 being different from those of amelogenin. Two other very minor protein components had amino acid compositions distinct from the amelogenins and the serum proteins, but were N-terminally blocked on attempted sequencing. None of the components in the neutral soluble low-ionic-strength extract or in the 4 M guanidine hydrochloride extract, both of which consist principally of amelogenins, immunoreacted with anti-albumin or with any of the antibodies to other serum proteins and fetuin, despite the fact that the amelogenin extracts also contain nonamelogenin proteins. On the basis of the data presented, studies employing antibodies to the so-called enamelin proteins and hypotheses as to their molecular conformation, their roles as evolutionary markers, or their positive role in mineralization should be reconsidered and reviewed. [ABSTRACT FROM AUTHOR]

Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5–37°C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r < 5) decreases with increasing temperature, whereas binding of the following molecules seems to proceed independently of temperature. Binding data were analyzed according to the general binding equation yielding several sets of acceptable binding constants within a probability limit of 0.75. From the temperature dependence of the first step constant, it was possible to calculate values for the changes in enthalpy and entropy during the initial binding step. For the medium-chain fatty acids, laurate and myristate, binding of the first molecule to albumin appeared to be enthalpic, with a tendency to an increasing contribution of entropy to binding energy with increasing chain length of the fatty acid. [ABSTRACT FROM AUTHOR]

Time-dependent NOE studies of the C13¹ and C17¹ methylene proton resonances of the heine peripheral propanoate groups have elucidated their mobility in the active site of the ferric high-spin form of Galeorhinus japonicus myoglobin. A large difference in the chemical shift due to the non-equivalence of the heine C13¹ and C17¹ methylene proton resonances allows selective irradiation of a given proton resonance by a high-power selective decouple pulse in spite of their fast relaxation rates. NOE accumulation of the resonance of one methylene proton after saturation of the resonance of the other proton essentially follows the theoretical prediction derived using the two-spin approximation, and the cross-relaxation rates for the heine C13¹ and C17¹ methylene proton spin systems were quantitatively determined. The correlation time for the mobility of the internuclear vector connecting the heine C13¹ or C17¹ methylene protons was then calculated from the cross-relaxation rate and values of approximately 11 ns were obtained for both C13¹ and C17¹ methylene groups in 2 mM Galeorhinus japonicus myoglobin at 35 °C. The immobile C13¹ and C17¹ methylenes of the heine propanoate groups, together with a large difference in chemical shift between the methylene proton resonances, dictate their fixed orientation with respect to the protein moiety as well as the heme plane, and are therefore consistent with the immobile heme in the active site of myoglobin. [ABSTRACT FROM AUTHOR]

Sulfotransferase, which catalyzes sulfation of the carbohydrate of galactosylceramide (GalCer) and is localised in the Golgi membrane of cells, was assayed for activity in human serum. To do this. an organic solvent was added to the incubated reaction mixture containing GalCer as an acceptor and phosphoadenosine phospho[35S]- sulfate as a donor of sulfate to dissociate the synthesized sulfolipid from serum protein. This was followed by isolation of the sulfolipid on an anion-exchange column. Through this procedure, human serum was found to contain sulfotransferase activity. The serum enzyme was activated by Mn2+, Km values of the enzyme for GalCer and ‘active sulfate’ were 4.6 μM and 5.2 μM, respectively. The enzyme activity was assayed in sera of cancer patients. The serum activity (mean ± SE, 0.27 ± 0.027 pmol · μl-1 · h-1) in renal cell carcinoma patients, whose activity has been demonstrated to be elevated, was significantly (P < 0.005) increased compared to that of the normal control (mean ± SE, 0.18 ± 0.0014 pmol · μl-1 · h-1) and of other urological tumors examined. [ABSTRACT FROM AUTHOR]

Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIll (30 U/ml) with 0.1 μg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity, in the presence of 10 μg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by yon Willebrand factor. At high concentrations of factor Xa (10 μg/ml), von-Willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of yon Willebrand factor and all fragments were released from yon Willebrand factor. The major products were light-chain-derived Fragments of molecular mass 66/68 kDa and 60 kDa and heavy-chain-derived fragments of 40 and 42 kDa. Also minor products of 12.20/21, 23, 27 and 30 kDa were observed, most of which were specific for cleavage of factor VIII by factor Xa. [ABSTRACT FROM AUTHOR]

The conditions for refolding reduced and denatured human serum albumin (HSA) were investigated with a view to maximising the yield of native monomeric albumin. Refolding by dialysis was found to be preferable to dilution as a means of chaotrope (urea) and reductant (2-mercaptoethanol) removal. Dialysis of denatured HSA solutions containing 4-8 M urea and 14 mM 2-mercaptoethanol at pH 10.0 was found to be optimal for HSA refolding. The yield of monomeric HSA was maximal (94%) for dialysis in the presence of EDTA (1 mM) and sodium palmitate (20 µM). Using this protocol it was possible to refold HSA at concentrations in excess of 5 mg · ml-1 whilst maintaining a high recovery of native monomer. These results represent a considerable improvement on established methods of HSA refolding. [ABSTRACT FROM AUTHOR]

Human inter-α-trypsin inhibitor is a plasma protein of MT 180000 which has long been described as a single polypeptide chain. However, we have previously demonstrated that it is synthesized in liver by two different mRNA populations coding for heavy or light polypepfide chains [Bourguignon, J. et al. (1983) FEBS Left. 162, 379-383] and cDNA clones for the heavy or light chains have recently been isolated and characterized [Bourguignon, J. et al. (1985) Biochem. Biophys. Res. Commun. 131, 1146-1153; Salier, J. P. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8272-8276]. In the present study, we show that human poly(A)-rich RNAs hybrid-selected with various heavy-chainencoding cDNA clones translate three different heavy chains, designated H1 (Mr 92000), H2 (Mr 98000) and H3 (Mr 107000). We previously characterized two heavy-chain cDNA clones. We now report that they correspond to H1 and H2 chains. We have also determined the sequence of an additional cDNA clone which codes for Ha chain. Its insert size is 1.79 kb with a single open reading frame and a poly(A) tail. The deduced amino acid sequence of the Ha chain is highly similar to those of the H1 (54%) and H2 (44%) chains. Northern analysis of human liver poly(A)-rich RNAs with the three heavy-chain cDNAs as probes clearly identified a single major mRNA population of 3.3 ± 0.1 kb. Chromosomal localization by in situ hybridization shows that inter-α-trypsin inhibitor genes are located on three different human chromosomes. The H1 and H3 genes are located in the p211 -p212 region of chromosome 3, whereas the H2 gene resides in the p15 band of chromosome 10. The light-chain gene is located in the q32- q33 region of chromosome 9. These results indicate that heavy and light chains of inter-α-trypsin inhibitor are encoded by at least four functional genes. [ABSTRACT FROM AUTHOR]

Using a radioimmunoassay for rabbit-serum albumin, platelet-activating-factor-induced serum-albumin release by platelets was monitored under non-aggregating conditions. The four main results from this study are as follows. The EC50 of the release was of the same order of magnitude as the aggregation EC50 in the presence of calcium. The release took place within 2 min and was inhibited by BN 52021, which is a very specific inhibitor of the platelet-activating-factor-aggregating effect. Serum-albumin release was much greater than serotonin release. [ABSTRACT FROM AUTHOR]

The hydrophobic compound diethylstilbestrol inhibits the generation of the proton gradient and the membrane potential in chromatophores from Rhodospirillum rubum and dissipates proton gradients over asolectin vesicle membranes. The Ca2+-ATPase activity of chromatophores, of purified F0F1-ATPase and of purified F1-ATPase is also decreased in the presence of diethylstilbestrol. Other repressed activities are the pyrophosphatase activity of soluble pyrophosphatase from yeast and the NADH oxidation by L-lactate:NAD oxidoreductase. We have previously reported that also ATP synthesis, PPi synthesis and PPi hydrolysis of R. rubrum chromatophores are inhibited by diethylsilbestrol [Strid et al. (1987) Biochim. Biophys. Acta 892, 236-244]. Addition of bovine serum albumin reverses or prevents diethylstilbestrol-induced inhibition of the activities tested. On the other band, the Mg2+-ATPase activity of chromatophores, purified F0F1-ATPase and purified F1-ATPase are stimulated by low concentrations of diethylstilbestrol. On the basis of its hydrophobicity and the reversal of its inhibition by bovine serum albumin, diethylstilbestrol is proposed to act unspecifically on membranes and at hydrophobic domains of proteins. Such an attack upon the subunits of the F1-ATPase, altering the subunit interactions, is proposed to explain the different results obtained for the Ca2+-ATPase and the Mg2+-ATPase. [ABSTRACT FROM AUTHOR]

Isolated β chains from human adult hemoglobin at millimolar concentration are mainly associated to form β4 tetramers. We were able to obtain relevant two-dimensional proton nuclear magnetic resonance (NMR) spectra of such supermolecular complexes (Mr &ap; 66000) in the carboxylated state. Analysis of the spectra enabled us to assign the major part of the proton resonances corresponding to the heme substituents. We also report assignments of proton resonances originating from 12 amino acid side chains mainly situated in the heme pocket. These results provide a basis for a comparative analysis of the tertiary heme structure in isolated β(CO) chains in solution and in β(CO) subunits of hemoglobin crystals. The two structures are generally similar. A significantly different position. closer to the heme center, is predicted by the NMR for Leu-141 (H19) in isolated β chains. Comparison of the assigned resonances of conserved amino acids in α chains, β chains and sperm whale myoglobin indicates a close similarity of the tertiary heme pocket structure in the three homologous proteins. Significant differences were noted on the distal heme side, at the position of Val-E11, and on Leu-H19 and Phe-G5 position on the proximal side. [ABSTRACT FROM AUTHOR]

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat α2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of α2macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, viceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of α2-macroglobulin expression are localized by in situ hybridization of cDNA for α2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for α2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal, α2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. α2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the α2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting α2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium. [ABSTRACT FROM AUTHOR]