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Reverse-phase HPLC was used to fractionate 40S ribosomal proteins from human placenta. Application of a C4 reverse-phase column allowed us to obtain 27 well-resolved peaks. The protein composition of each chromatographic traction was established by two-dimensional polyacrylamide gel electrophoresis and N-terminal sequencing. N-terminally blocked proteins were cleaved with endoproteinase Lys-C, and suitable peptides were sequenced. All sequences were compared with those of ribosomal proteins avail- able from data bases. This allowed us to identify all proteins from the 40S human ribosomal subunit in the HPLC elution profile. By matrix-assisted laser-desorption ionization mass spectrometry the masses of the 40S proteins were determined and checked for the presence of post-translational modifications. For several proteins differences to the deduced sequences and the calculated masses were found to be due to post-translational modifications. [ABSTRACT FROM AUTHOR]

Aryl acylamidase was purified from an extract of N-acetyl-o-toluidine-induced cells of Nocardia globerula IFO 13510 in ten steps. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of approximately 126 kDa and consists of two subunits which are identical in molecular mass. The purified enzyme catalyzed the hydrolysis of N-acetyl-o-toluidine to o-toluidine and acetic acid at a rate of 47.7 µmol · min-1 · mg-1 at 35°C. It also catalyzed the hydrolysis of various anilide derivatives and esters, as well as the transfer of an acetyl group to aniline as an acetyl acceptor. The purified enzyme was sensitive to thiol reagents such as HgCl2 and p-chtoromercuribenzoate. The amino-terminal sequence (28 amino acid residues) of the enzyme was determined. Based on the substrate specificity of this enzyme, the pathway intermediates involved in the conversion of n-acetyl-o-toluidine to 4'-hydroxy-N-acetyl-o-toluidine are discussed. [ABSTRACT FROM AUTHOR]

A plasminogen activator inhibitor (PAI) was purified from bovine endothelial cell conditioned medium by a simple procedure in the absence of protein denaturant. The yield was 2.2 mg from 1.61 conditioned medium in a typical experiment. The purified inhibitor showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and reverse fibrin autography with an apparent molecular mass of 45 Wa. The amino-terminal 40-amino-acid sequence was determined and found to be 70% similar to the reported corresponding sequence of human PAI-1. The amino acid composition also revealed a close relationship between bovine PAI and human PAI-1. The purified PAI was substantially inactive (570 U/mg) but it could be activated by treatment with protein denaturants such as 1% SDS (1.8 × 105 U/mg) and 4 M guanidine-HCl (1.5 × 105 U/mg). A more effective activation of this latent PAI was achieved by heat treatment at 100°C for 2.5 min, generating the specific activity of 1.0 × 106 U/mg. The heat-activated PAI lost its activity during incubation at 56°C for 30 min, but repeated heat at 100°C for 2.5 min could regenerate about 70% of the initial activity. Treatment at 37°C, 56°C and 80°C. however, failed to activate the latent PAI at all. These findings suggest that the buried reactive site of the latent PAI is exposed as a result of a heat-induced, specific conformational change, but tends to be masked again during renaturation under mild conditions, i.e. the PAI protein takes on preferentially a latent form. [ABSTRACT FROM AUTHOR]

The effects of heat treatment of CV1 cells infected with simian virus 40 (SV40) on viral and cellular protein synthesis were investigated by one-dimensional and two-dimensional polyacrylamide gel electrophoresis. A 12-h heat treatment during the late phase of the viral life-cycle inhibits VP1 synthesis. No inhibition of normal cellular proteins is apparent, but heat-shock proteins are strongly induced and accumulate in the cells. Inhibition of VP1 synthesis in infected cells is demonstrated to occur also after arsenite treatment, another agent known to induce heat-shock proteins. Northern blot analysis of cytoplasmic RNA demonstrated a decrease in the abundance of late SV40 mRNAs thus showing that the inhibition occurs at the transcriptional or immediately post-transcriptional level. Cumulative labeling with [³H]thymidine of viral DNA showed that the decrease in the abundance of late mRNAs is not due to a blocking of viral DNA synthesis. Immunofluorescence microscopy and immunoprecipitation analysis show that heat and arsenite treatments also affect the synthesis of T antigen. These results suggest that heat-shock proteins may play a role in the inhibition of SV40 virus gene functions. [ABSTRACT FROM AUTHOR]

A new acidic form of glutathione S-transferase (OST, p1 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (Mr or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the Mt in isoelectric point, activity towards I ,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn2. The activity was inhibited by sulfasalazine, an inhibitor of leukotrienc-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 μM and 3.5 mM respectively. A major OST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotrienc A4 was 100 gM, fourfold higher than that of GST-Yn1Yn1. These results suggest that mainly GST-Yn1Yn1 may be involved in leukotriene-C4 synthesis in rat brain. [ABSTRACT FROM AUTHOR]

Rabbit α1-microglobulin was purified from the urine of sodium-chromate-treated animals by the use of gel chromatography on Septiadex G-100, affinity chromatography on concanavalin-A - Sepharose and ion-exchange chromatography on DEAE-Sepbadex. Rabbit α1-microglobulin had a molecular mass of 25.6 kDa on SDS/ polyacrylamide gel etectrophoresis. α1-microglobulin has previously been purified from the urine of humans, guinea-pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of α1-microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human α1-microglobulin sample, and only two bands in guinea-pig, rat and rabbit α1-microglobulin. with a gap between each band of 2.6 - 2.9 kDa. The amino-terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino-terminal amino acids present in the other species were missing in guinea-pig α1-microglobulin. Our results indicate that human α1-microglobulin is substituted with two N-linked oligosaccharides, white only one is attached to each of the other α1,-microglobulins, and that the extra glycosylamine-linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the Nelinked oligosaccharides carry the immunosuppressive activity of α1-niicroglobulin. [ABSTRACT FROM AUTHOR]

A novel assay has been developed for 2-deoxy-2-sulphamido-D-glucose (GlcNS) sulphamidase from Flavobacterium heparinum. This has enabled the 1930-fold purification of the enzyme from a soluble fraction of bacterial homogenate. From SDS/polyacrylamide gel electrophoresis the enzyme was shown to have a relative molecular mass of 81 500. Ca2+ was essential for enzyme activity. Inorganic phosphate and sulphate inhibited activity by 28% and 29% respectively at 5 mmol dm-3. The purified sulphamidase had a pH optimum of 7.0 and a Km of 8.32 µmol dm-3 for GlcNS. The degradation of 2-deoxy-2-sulphamido-6-O-sulpho-D-glucose (GlcNS-6S) was also re-investigated. The two sulphate groups were hydrolysed sequentially in a single non-bifurcate manner, in contrast to previous reports [Dietrich, C. P., Silva, M. E. and Michelacci, Y. M. (1973) J. Biol. Chem. 248, 6408-6415]. [ABSTRACT FROM AUTHOR]

Complexes containing rat liver SOS ribosomes treated with puromycin and high concentrations of KC1, elongation factor 2 (EF-2) from pig liver, and guanosine 5'[β,y-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-inked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by auto radiography. The following proteins of both large and small subunits were identified: L9, LU, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/ S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33 -L11 and LA33-L12. [ABSTRACT FROM AUTHOR]

When a 100000 x g supernatant from bovine heart was incubated with [1-14C]eleic acid ad subjected to isoelectric focusing, two fatty acid proteins (FABPS) with isoelectric points 4.9 and 5.1 were detected. The proteins were purified in a large acid precipitation of a postmitochondrial supernatant, as well as fractionation with ammonium sulfate, then by alternate application of ion-exchange and gel chromatography. The procedure afforded around 60 mg pure proteins from 1.5 kg fresh heart muscle. Relative molecular masses of 15300 ± 1600 for both proteins were derived from sodium dodecyl sulfate/polyacrylamide gel electrophorosis, gel chromatography, sedimentation velocity as well as from amino acid analysis. Up to 50% of the proteins secondary structures consisted of β-sheet. N-termini of the peptide chains were blocked; the amino acid compositions of the two proteins were similar, but differed considerably from those of the two FABPs isolated from bovine live [Haunerland et al. (1984) Hoppe Seyler's Z. Physiol. Chem. 365, 365-376]. Whereas hepatic FABPs changed their p1 upon binding fatty acids, cardiac FABPs did not. Cardiac FABPs were immunologically identical, but did not cross-react with hepatic proteins. A reversible, concentration-dependent self-association reported for FABP from pig heart [Fournier et al. (1983) Biochemistry 22, 1863-1872] was not observed for FABP from bovine heart. Changes of concentration did not alter secondary structure, intrinsic fluorescence or the sedimentation coefficient of the protein. [ABSTRACT FROM AUTHOR]

Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in ≈ 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170000 under nonreducing conditions and 135000 (α subunit) and 25000 (β subunit) under reducing conditions. GP IX had an apparent Mr of 22000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ibα had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib, and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ibα and GP IX were blocked; GP Ibβ had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (&xsline; ± SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ibα (22000 ± 2700, n = 3), WM 23, epitope on GP Ibα (21000 ± 3400, n = 3), FMC 25, epitope on GP IX (20100 ± 2700, n = 3), and FMC 25 (Fab')2 (27100 ± 800, n = 2). The combined evidence suggests that GP Ib is normally bound to GP IX and that the stoichiometry of this complex is 1 : 1. [ABSTRACT FROM AUTHOR]

Highly purified peptide elongation factor 1 from rabbit reticulocytes liberates the terminal phosphate from [γ-32P]GTP and incorporates it into its own protein. Approximately one phosphate residue becomes bound by one molecule of the factor. Only the eEF-1α subunit of the factor (Mr 53 000) becomes phosphorylated as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by autoradiography and by the incubation of [γ-32P]GTP with individual subunits of the elongation factor separated by chromatofocusing in the presence of 5 M urea. The phosphorylation also takes place, though to a lesser extent, if the factor is incubated with Na2H32PO4, probably due to the presence of endogenous GTP bound in the molecule of the factor. The content of endogenous GTP in various factor preparations was 0.21-0.43 mol/mol factor. Phosphorylation of the peptide elongation factor is ribosome-independent, acid-labile and apparently autocatalytic since no other proteins are required for this reaction. Preincubation of the factor with GTP or with inorganic phosphate results in the phosphorylation of the factor and is followed by an enhanced binding of phenylalanyl-tRNA to 80S ribosomes in the presence of poly(U). This is accompanied by a dephosphorylation of the factor protein and thus the reversible autophosphorylation of the factor apparently activates its binding site for aminoacyl-tRNA. This is supported by the observation that sodium fluoride, which inhibits the dephosphorylation of the factor, blocks the factor-catalyzed binding of aminoacyl-tRNA to ribosomes. The incorporation of phosphate into factor protein also inhibits the formation of an eEF-1 · GDP complex, which is inactive in protein synthesis. Thus GDP liberated by the GTPase activity of the factor cannot affect its binding site for aminoacyl-tRNA. This may be the other reason for the enhanced activity of the phosphorylated factor. The autocatalytic GTP-dependent phosphorylation of the peptide elongation factor 1 apparently modifies its function and may thus play a regulatory role in protein synthesis. [ABSTRACT FROM AUTHOR]

Analysis by crossed-immunoelectrophoresis of Paracoccus denitrificans membrane vesicles has shown that only one antigen stains for NADH dehydrogenase activity. This activity could be partially purified by a combination of gel filtration and ion-exchange chromatography of membrane vesicles that had been solubilised in the non-ionic detergent Nonidet P-40. From the limited number of precipitates observed after crossed immunoelectrophoresis of this partially purified preparation of NADH dehydrogenase it was possible to excise specifically part of the precipitate that stained for NADH dehydrogenase. Excised precipitates containing NADH dehydrogenase that had been radiolabelled by growth of cells in the presence of [35S]SO42- allowed the polypeptide composition of the enzyme to be determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by fluorography. Two subunits were identified with estimated relative molecular masses of 48 000 and 25 000. Subunits of similar molecular weight are found in the flavoprotein fragment of the NADH dehydrogenase of the mammalian mitochondrial respiratory chain. The latter has general similarities with the respiratory chain in the plasma membrane of P. denitrificans. [ABSTRACT FROM AUTHOR]

Proteins L6 and L29 occupy closely adjacent sites in mammalian 60-S ribosomal subparticles and are easily crosslinked by intermolecular disulfide bond formation. For locating the interacting thiols within the polypeptide chains the dissociated proteins L6 and L29 obtained from the isolated disulfide complex wore subjected to S-cleavage following [14C]cyanylation of the two cysteine residues. Four split products of the [14C]cyanylated proteins were isolated by dodecylsulfate gel electrophoresis. Two of those could be identified by autoradiography as the selectively labeled Cterminal fragments. For unequivocal assignment of the fragments to the parent proteins, a simple and generally applicable method of cleaving cyanylated protiens in polyacrylamide gel for subsequent diagonal analysis was developed. The experiments indicated that the sulfhydryl group of L6 interacting with L29 is located at a distance of approximately 80 amino acid residues from the N-terminus. In the intact ribosome this sequence contains a clostripain-sensitive and trypsin-sensitive portion of the protein more or less exposed at the ribosomal surface. In the case of protein L29, the interacting sulfhydryl group was located at a distance of approximately 40 amino acid residues from the C-terminal. [ABSTRACT FROM AUTHOR]

In purified solutions of α-actinin from human blood platelets, three polypeptides a,b and c, of approximately 100 kDa, were observed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were identified as α-actinin subunits on the basis of their cross-reactivity with antibodies against skeletal muscle α-actinin and their interaction with F-actin. After electrophoresis in the absence of sodium dodecyl sulfate, six forms were observed: aa, ab, bb, ac, bc, cc. The similarity between the one-dimensional peptide maps of a and b and the absence of b in the presence of calcium-dependent protease inhibitors indicated that his probably derived from the a subunit. The c subunit differs from the a subunit. The results provide evidence that there are actually only two platelet α-actinin subunis a and c which give rise to three isoforms: two homodimers aa and cc, and one heterodimer ac. [ABSTRACT FROM AUTHOR]

Poly(A)-protein particles were prepared from rat liver nuclear extract after digestion with pancreatic ribonuclease and ribonuclase T1 by sucrose gradient centrifugation. The particles were sedimented in a range of 9-23 S with a peak at 16S. The particles isolated in this manner were 99- 100% resistant to further pancreatic ribonuclease treatment and contained more than 90 % adenylic acid, In CsC1 density gradient the nuclear poly(A)-protein particles banded in a narrowdensity rangeof 1.28- 1.32g/cm3 with a peak at L30g/cm3, whichcorrespondsto about 90%,of protein in the particies. The average length of the poly(A) molecules prepared from the 16-S particles was about 140 nucleotides. Urea/sodium dodecyl suiphate/polyacrylamide gel electrophoresis demonstrated two major polypeptide components with Mr of 63 000 and 90000 and at least ten minor polypeptides in the 45000- 130 000-Mr range. In sodium dodecyl suiphate/polyacrylamide gels the 63000-Mr polypeptide was the only one major component. Amino acid analysis of the polypeptides bound to nuclear poly(A) revealed that the polypeptides contained a relatively large amount of aspartic acid + asparagine and glutamic acid + glutamine (24%). Treatment of glutaraldehyde-fixed particles with micrococcal nuclease showed that more than 90 % of the poly(A) was accessible to the enzyme. thus almost the entire poly(A) should be located on the surface of the particles. On the basis of the results a model for the 'average' 16-S particle was constructed. [ABSTRACT FROM AUTHOR]

The large bacteriochlorophyll-a-binding polypeptide of the light-harvesting complex II (B800-850), having an apparent Mr with sodium dodecyl sulfate/polyacrylamide electrophoresis of 10000, has been isolated and purified from intracytoplasmic membranes of the phototrophically negative mutant strain Y5 of Rhodopseudomonas capsulata. The primary structure of this polypeptide has been determined. The polypeptide consists of 60 amino acid residues yielding an Mr of 7322. The hydrophobic stretch in positions 16-35 with a histidine in position 31 might be of importance for interaction with bacteriochlorophyll. The C-terminal part is also hydrophobic while the N-terminal part consists of hydrophilic amine acids. The polarity of the total amino acids was determined to be 28.3%. [ABSTRACT FROM AUTHOR]

Preulbumin was purified from rat plasma by chromatography on Blue Sepharose CL-6B, followed by chromatography on concanavalin-A—Sepharose and preparative electrophoresis in polyacrylamide gel. The overall recovery was 20%. The purified prealbumin was homogeneous upon electrophoresis in detergent containing polyacrylamide gel and was used to raise a monospecific anti-serum in rabbits. During perfusion of rat liver, [14C]-leucine was incorporated into prealbumin secreted into the perfusion medium, suggesting that the liver was synthesizing prealbumin. The ratio of the rate of incorporation of leucine into prealbumin to that into total protein was 1.5%. 125I-prealbumin had a half-life of 29 h in the bloodstream of healthy Buffalo rats on a diet containing 20% protein. Whole body homogenates from healthy rats contained 6.2 mg prealbumin/100 g body weight. The rate of synthesis of prealbumin calculated from half-life and total body pool, was 3.6 mg prealbumin ×(100 g body weight)-1 × day-1. Two days after induction of inflammation by a subcutaneous injection of turpentine, both the concentration of prealbumin in the plasma and the amount of prealbumin in the whole body homogenates decreased to a minimum of about one third of normal. The relative rate of degradation of 125I-prealbumin did not change during inflammation. The rate of synthesis of prealbumin decreased considerably, or even ceased, during acute inflammation. [ABSTRACT FROM AUTHOR]

Tryptophan 5-monooxygenase was purified 880-fold with a 48% yield from mouse mastocytoma cells (P815) by only a one-step purification procedure of pteridine affinity chromatography. The specific activity of the final preparation was 5280 nmol min-1 mg-1. It gave a single protein band on polyacrylamide gel electrophoresis in the absence and presence of sodium dodecylsulfate. The molecular weight of the enzyme was determined to be 270000 by gel filtration and 280000 by gradient polyacrylamide gel electrophoresis. Sodium dodecylsulfate/ polyacrylamide gel electrophoresis revealed the enzyme to be composed of identical subunits with a molecular weight of 53000. Tetrameric structure of the enzyme was suggested by cross-linking studies using dimethyl suberimidate as a bifunctional reagent. The isoelectric point of the enzyme was estimated to be 6.0. Amino acid analysis showed a residue composition similar to that reported for rat liver phenylalanine 4-monooxygenase. The enzyme activity was stimulated approximately fivefold by preincubation with dithiothreitol and Fe2+. The purified enzyme had an activity of phenylalanine hydroxylation and also a weak activity of tyrosine hydroxylation. The kinetic properties of the enzyme are also presented. [ABSTRACT FROM AUTHOR]

Two acidic proteases from the white-rot fungus Sporotrichum pulverulentum have been purified and partially characterized. The enzymes were purified in four steps, protease I 152-fold and protease II 127-fold. The purity of the enzymes was investigated by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis. The isoelectric points of protease I and protease II were at pH 4.7 and 4,2 respectively. The molecular weights were 28000 and 26000 and the pH optima 5.0 and 5.2. Both enzymes were inhibited by heavy metal ions such as Ag+, Hg+ and to some extent also by Cu2+. Partial inhibition was also observed with EDTA and α,α'- dipyridyl. The specificities of protease I and II were investigated using human fibrinopeptide A as substrate. Protease I splits off the arginine in the C-terminal position, while protease II splits at the carboxyl side of both Phe-8 and Leu-9 in fibrinopeptide A. A physiological effect of the two proteases has also been demonstrated. Thus, if a culture solution from S. pulverulentum grown on cellulose was treated with the individual proteases or a mixture of the enzymes a considerable increase in endo-1 ,4-β-glucanase activity was obtained. It may be that the endo-glucanases are produced in a zymogenic form and activated by the proteases. However, other explanations for the phenomenon are also possible. [ABSTRACT FROM AUTHOR]

Polyacrylamide gel electrophoresis is one of the most efficient methods for separation and identification of proteins. A new gel electrophoresis system has been developed in order to facilitate both separation and extraction of peptides and proteins ranging in molecular weight from approximately 200 up to 100000. This system involves the use of a volatile buffer. triethylamine/formic acid pH 11.7, and a reaction with a covalently binding NH2 reagent. I ,3,6-trisuifonylpyrene 8-isothiocyanate. Under these conditions, the addition of strongly negative charges to proteins is achieved which allows migration according to molecular weight. The modified proteins being fluorescent require neither fixation nor staining for their detection. This is an absolute requirement especially when dealing with small peptides. An additional advantage of such a modification is the increased solubility of the proteins which makes their extraction easier and more efficient. The extracted proteins are easily freed from salts and other small molecules simply by evaporation and they are readily available for sequencing analysis using Edman degradation. carboxypeptidase digestion or amino acid analysis. [ABSTRACT FROM AUTHOR]

A specific type-II restriction endonuclease BcnI from Bacillus centrosporus has been purified to electrophoretic homogeneity in three chromatographic steps. Around 15 μg of such a preparation can be isolated from 1 g of the cell paste. The yield of the enzyme is higher than that of any type-II restriction endonuclease so far reported. The molecular weight of the enzyme determined by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate equals 27500 and 28000 respectively. The activity of the restriction endonuclease is maximal at pH 9.2 and 40 -45 °C. The optimal magnesium concentration was estimated to be 7.5 mM. The activity of BcnI may also be observed in the presence of Co2+, Mn2+, Ni2+ and Zn2+ but it is markedly less than in the presence of Mg2+. [ABSTRACT FROM AUTHOR]

Examines the binding of the 125 I-labeled lectin to various erythrocytes. Polyacrylamide gel electrophoresis; Molecular weight; Dissociation of the lectin; Purification of vicia graminea lectin.

The order in which ribosomal proteins become associated with nucleolar pre-rRNA was studied by measuring the kinetics of label accumulation in ribosornal proteins isolated from the cytoplasmic ribosomes, as well as by two-dimensional polyacrylamide gel electrophoresis of protein extracted from ribosorne precursor particles. Tentative identifications of early-adding and late-adding groups of proteins have been made. [ABSTRACT FROM AUTHOR]

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. We detect no differences between the fixation of E. coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per α2ββ′ unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per α2ββ′ unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist. Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase. [ABSTRACT FROM AUTHOR]

Activity of pseudocholinesterase (acylcholine-acyl-hydrolase) elevated up to four times has been detected in sera of members of two German families. The catalytic concentrations of the pseudocholin- esterase of the afflicted members of both families (male and female) varied between 4800 U/I and 10200 U/I (acetylthiocholine iodide substrate). The pseudocholinesterase of the propositi exhibits isoenzyme separation patterns in polyacrylamide electrophoresis as well as in electrofocussing which are different from those of pseudocholinesterase from normal persons. No differences could be seen as regards the Km of substrates or the inhibition by dibucaine, fluoride or succinyldiocholine. [ABSTRACT FROM AUTHOR]

8-Azido-adenosine 5'-triphosphate (n38ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated fonn of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n38ATP incorporated into the holoenzyme is covalently bound upon irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n38ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [γ32] n38ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site. [ABSTRACT FROM AUTHOR]

Three groups of proteins can be clearly discriminated in the total protein of L cell polysomes by selective labelling in the presence of low doses of actinomycin D and two-dimensional polyacrylamide/ dodecylsulfate gel electrophoresis followed by autoradiography: (a) structural ribosomal proteins which are not labelled in the presence of actinomycin D and form stained non-radioactive spots in gels; (b) exchangeable ribosomal proteins which are labelled in the presence of actinomycin D and form stained radioactive spots; (c) non-ribosomal proteins which are detectable only by autoradiography of gels. The large and small subunits of L cell ribosomes contain respectively 45 and 34 ribosomal proteins with molecular weights ≤ 50000; seven of the large subunit proteins and nine of the small subunit proteins are exchangeable. Most of the non-ribosomal proteins migrate in the region of the second-dimension gel slab corresponding to the molecular weight range 50000-120000. Problems related to the separation of the ribosomal proteins of mammalian cells and the possible significance of the presence of non-ribosomal proteins in polysomes are discussed. [ABSTRACT FROM AUTHOR]

A method is described for the large-scale purification of membrane fragments very rich in acetylcholine (nicotinic) receptor from the electric organ of Torpedo marmorata. The preparations of purified membrane fragments have a specific activity of more than 4000 nmol α-toxin binding sites/g protein and give only four main polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Observations by electron microscopy show that the purified preparation of receptor-rich membrane fragments is composed of only one class of membrane fragments covered with 8-nm rosettes identified as acetylcholine receptor molecules. This preparation is used as a starting material for the detergent solubilization and the large-scale purification of the acetylcholine receptor protein, without using affinity chromatography. A sucrose gradient centrifugation of a Triton X-100 extract of receptor-rich membranes done in the presence of 2-mercaptoethanol yields large quantities of receptor protein in a homogeneous form as indicated by polyacrylamide gel electrophoresis, isoelectric focussing and electron microscopy. Polyacrylamide gel electrophoresis of the purified protein in the presence of sodium dodecyl sulfate reveals three bands of apparent molecular weights 40000±1000, 50000 ± 2000 and 66 000 ± 2000, the two heaviest ones being present in significantly lower amounts than the 40 000-M1 one. The comparison of several samples of purified protein with different specific activities reveals a striking variability of the ratios of the 40000- M1, band to the 50000 and 66000- M1 ones. [ABSTRACT FROM AUTHOR]

Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. The enzyme has a molecular weight of about 200000 and is a hornopolymeric assemblage of either three or four subunits of molecular weight 54000–58000. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein. The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixed-function oxygenases with external electron donors. The oxygenated product has been identified as 1-oxa-2-oxocyclohexane (5-valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2-lactouizing) (EC 1.14.13.—). Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p-hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent. The enzyme has a pH optimum of 7.7. The Km, for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits. Anaerobic reduction of enzyme-bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments. Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated. [ABSTRACT FROM AUTHOR]

1. Two mitochondrial forms of acetoacetyl-CoA thiolases designated as enzyme A and enzyme B were crystallized from ox liver. They could be shown to be homogenous by polyacrylamide gel electrophoresis. 2. In direction of acetoacetyl-CoA cleavage enzyme A shows a double competitive substrate inhibition when acetoacetyl-CoA is varied at different fixed CoA concentrations. With enzyme B a parallel kinetic pattern is obtained when acetoacetyl-CoA is varied at different fixed CoA concentrations. In direction of acetoacetyl-CoA synthesis both enzymes show linear reciprocal plots of initial velocities against acetyl-CoA concentrations in absence of CoA. These initial velocity kinetics in the forward and in the reverse direction are in accordance with a ping-pong mechanism of reaction for both enzymes involving an acetyl-S-enzyme as intermediate. 3. Under saturating concentrations of substrate, the ratios of acetoacetyl-CoA synthesis/aceto-acetyl-CoA cleavage is 0.31 for enzyme A and 0.08 for enzyme B. The maximum velocity in direction of acetoacetyl-CoA synthesis of enzymes A and B are 0.43 μmol × min-1 × unit thiolase-1 and 0.10 μmol × min-1 × unit thiolase-1, respectively. 4. Both enzymes show nearly the same affinity for acetyl-CoA. The Km values are 91 μM (enzyme A) and 80 μM (enzyme B). 5. Coenzyme A and acetoacetyl-CoA both act as inhibitors in direction of acetoacetyl-CoA synthesis: coenzyme A is a nonlinear competitive inhibitor of both enzymes. Acetoacetyl-CoA exerts a negative cooperativity on enzyme A (nH = 0.63) and is a competitive inhibitor for enzyme B (Ki = 1.6 μM). 6. The catalytic and regulatory properties of the acetoacetyl-CoA thiolases A and B are discussed in terms of their proposed role in regulating ketogenesis. Intracellular fluctuations of acetoacetyl-CoA/3-hydroxybutyryl-CoA ratios, resulting in a suspension of inhibition of both enzymes at high NADH/NAD ratios, are postulated as a control mechanism of ketogenesis in addition to mechanisms already known. [ABSTRACT FROM AUTHOR]

A two-dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane. Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate—polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent-free polyacrylamide gradient gel. By this method the proteins of the human erythrocyte membrane could be resolved into a two-dimensional pattern, which has been shown to be highly reproducible with respect to various blood-groups and within one blood-group from specimen to specimen. The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III [Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606–2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrane. The two spectrin bands differed considerably in their two-dimensional patterns. The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia. [ABSTRACT FROM AUTHOR]

For chemical characterization of the products of activated lymphocytes a radioactive double-label technique was developed which allows one to distinguish those products synthesized either de novo or in increased amounts by the stimulated culture. Spleen cells from Balb/c mice were cultured in serum-free medium in the presence or absence of concanavalin A and simultaneously labelled with radioactive leucine. Optimal culture conditions were established by determining parameters such as cell density, mitogen concentration, and kinetics of protein synthesis following stimulation. Combined supernatants of stimulated and unstimulated cultures each labelled with either [³H]leucine or [4C]leucine were fractionated on Sephadex G-75. Materials derived from control or stimulated supernatants both yielded a qualitatively similar radiolabelled profile. The isotope ratio of stimulated to nonstimulated culture, however, showed a broad peak at KD 0–0.35 (approx. mol. wt 75000–20000) which was further analyzed by isoelectric focusing. Pools of every two fractions were focused in polyacrylamide gels at pH 3.5–10. By determining the isotope ratio, the isoelectric point, and the KD (mol wt), it was possible to distinguish at least 24 molecules which had been produced only, or in greater degree, by the stimulated culture. [ABSTRACT FROM AUTHOR]

Calcitonin biosynthesis has been studied in chicken ultimobranchial glands incubated in vitro in the presence of radioactive amino acids. The results obtained suggest the existence of a biosynthetic precursor of higher molecular weight or procalcitonin. This precursor has been identified by pulse-chase experiments, molecular weight determinations, biological activity measurements and analysis of tryptic peptides. Its molecular weight is about 13000 (calcitonin, about 3500) as determined by polyacrylamide gel electrophoresis. Procalcitonin is present in small amounts in chicken ultimobranchial glands and it is biologically active in rats. [ABSTRACT FROM AUTHOR]

The chorionic somatomammotropic hormone extracted from the human placenta exists in several molecular forms. Analytical electrophoresis in polyacrylamide gel permits separation of a highly anodic migration form, form 1, and another form migrating slightly faster than albumin, form 2. These two forms are active as measured by radioactive immunological analysis, form 2 being about 25 times more active than form 1. The two forms are mutually interconvertible. The two forms may also be separated by filtration on Sephadex G-50. However, they do not differ in molecular weight, they have the same coefficient of sedimentation and the same coefficient of apparent diffusion, measured by analytic ultracentrifugation. Glutaraldehyde and 8 M urea do not modify their electrophoretic or chromatographic behaviour. On the other hand, the two forms differ in their tertiary structure, with the modification depending on the greater or lesser degree of oxidation of the intra-chain disulfide groups. The two forms also exist in placental culture media and the incorporation of tritiated leucine occurs preferably in form 1. The physiological significance of the two hormone pools is not clarified. [ABSTRACT FROM AUTHOR]

New methods are presented for the isolation of nucleoli and the extraction of intact preribosomes under mild conditions avoiding EDTA and high salt buffers. These nucleolar preribosomes were characterized by an improved technique of polyacrylamide-gel electrophoresis which loves better resolution than sedimentation analysis for both ribonucleoprotein particles anti free-RNA molecules. Thus, the 50-S native-ribosomal subunit could be separated from its 55-S precursor containing 32-S pre-RNA, and another nucleolar precursor, a "70-S" particle, could be identified. The separation technique newly developed is disc electrophoresis using exponential polyacry1amide gels. The resolution with these gels is gloater than that with uniform gels over a wider range of molecular weights. The final resolution is independent of the volume in which the sample is loaded and the diffusion of molecules during migration is eliminated. Molecular weights of RNA molecules can be estimated in exponential gels as in uniform gels. Electrophoresis in quartz tubes allows the direct scanning of ultraviolet, absorbance during and after the run. As all handling is thus avoided, gels of very low concentration can be used. [ABSTRACT FROM AUTHOR]

All 21 ribosomal proteins from 30 S subunits of Escherichia coli were isolated in a pure state by the following methods: separation of subunits by zonal centrifugation in B XV rotors, extraction of proteins, CM-cellulose chromatography with pyridine formate gradients, gel filtration on Sephadex G-100 and, in some cases, preparative polyacrylamide block electrophoresis. The purity of isolated proteins was 97-100% shown by disc electrophoresis at pH 4.5 in urea, by two-dimensional polyacrylamide electrophoresis, by dodecylsulfate gel electrophoresis and by cellulose acetate gel electrophoresis. The yield of the isolated proteins was 7-110 mg depending on the protein and the number of purification steps. Fractionation of total 30 S proteins with ammonium sulfate is recommended for quick isolation of proteins S15 and S20 in large quantities. [ABSTRACT FROM AUTHOR]

The viscosity of histone-depleted nucleohistone has been measured at high and low ionic strength. Native nucleohistone at low ionic strength and f1-depleted nucleohistone at both low and high ionic strength all have the same intrinsic viscosity of 10.0 dl/g. This implies that these molecules are not flexible but behave in solution as rigid particles. On the assumption that they are rods they have a weight-average length of 1160 ± 120 nm and diameter 5.6 ± 0.3 nm. The DNA is compressed in the rod to give an increase in mass per unit length for the DNA of 5.0 ± 0.5. By contrast the intrinsic viscosity of nucleohistone depleted of f1, f2a2 and 75% of F2b and F3, varies with ionic strength in the manner typical of a flexible polyelectrolyte. The shapes of the sedimentation boundaries measured at high ionic strength of depleted nucleohistone show that the dissociation of histone from DNA takes place so that all molecules are depleted to the same extent. It thus has a conformation intermediate between that of native nucleohistone and DNA. Polyacrylamide gel electrophoresis of histones removed by 1.0 M NaCl using a technique capable of resolving all five main histone fractions, shows that if one fraction only is responsible for supercoiling nucleohistone it must be f2a2. [ABSTRACT FROM AUTHOR]

The lysyl ribonucleic acid synthetase from Escherichia coli has been purified to apparent homogeneity as judged from polyacrylamide gel electrophoresis and sedimentation velocity centrifugation. A molecular weight of 100,000 has been calculated for the enzyme. [ABSTRACT FROM AUTHOR]

The major parvalbumin from the white muscles of the hake (Merluccius merluccius) has been isolated in gram quantities in a state of homogeneity as evidenced by disc electrophoresis in 12% polyacrylamide gel at pH 9.4. by cellulose-acetate electrophoresis at pH 5.5, by sedimentation and by diffusion. Its hydrodynamic properties (D = 14.8 x 10-7: cm² x sec-1, s = 1 .79 x 10-13 sec. M = 11 500) and spectral characteristics (ε259 nm = 2.02 X 10² M-1) have been investigated, and its aminoacid composition determined as being Lys12, His, Arg, Asx12-13, Thr5, Ser5, Glx10, Gly12, Ala19, ½ Cys, Val4, Met, Ile7, Leu8, Phe10, with 3 amide groups and i free sulfhydryl. The protein has been shown to be free of any other constituent except for the presence of about o mol Ca/mol protein. No free X-terminus could be detected, but glycine was found to be C-terminal. The significance of these findings is discussed, and, in particular, possible similarities between parvalbumins and troponin A are explored from a functional and evolutionary point of view. [ABSTRACT FROM AUTHOR]

Proteose-peptone component 8F (or ‘8-fast’) has been prepared from bovine milk. Sedimentation equilibrium analysis, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and gel filtration in urea-containing buffers all gave molecular weight values between 3300 and 3900. The N-terminal sequence was found to be Arg-Glu- by dansylation and Edman degradation. Hydrazinolysis released lysine from the C-terminus. A mixture of carboxypeptidases A and B showed that the C-terminal sequence was -Thr-(Arg,Ile,Asn)-Lys. The phosphate content was 3.8 mol/mol and was completely released by a short alkaline hydrolysis indicating linkage to serine. This and all other aspects of the composition were entirely consistent with the identification of this proteose- peptone as residues 1–28 of the β-casein molecule. This identity was confirmed by a peptide mapping procedure. Thus proteose-peptone component 8F represents the N-terminal fragment when the γ1-caseins are formed by proteolysis of β-casein. [ABSTRACT FROM AUTHOR]