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The ratio of Ggamma to Agamma was studied in 13 normal healthy newborns and in eight neonates with hydrops fetalis due to homozygous alpha-thalassemia. The findings in the normal healthy newborns agreed with those of earlier reports. In homozygous alpha-thalassemia the Ggamma and Agamma ratio appeared generally lower than in healthy control newborns, but one of the hydrops fetalis cases had a very high Ggamma value. In all 13 normal healthy newborns, in 8 patients with homozygous alpha-thalassemia, and in 4 patients with homozygous beta-thalassemia, Tgamma chains with threonine at position 75 were detected in addition Igamma chains with isoleucine at position 75. In homozygous alpha-thalassemia, the Tgamma-to-Igamma ratio seemed lower and in homozygous beta-thalassemia higher than in normal newborns.

The amount of antigen on the solid phase was studied during various steps of an enzyme-linked immunosorbent assay (ELISA) for chicken anti-bovine serum albumin (BSA) antibodies. Three materials for the solid phase were used: polystyrene, nylon and cyanogen bromide activated paper. There was noticeable leakage of antigen during the assay from both polystyrene (30%) and nylon (60%). This occurred during all steps of the assay: during incubation with the sample serum, incubation with the enzyme conjugated antibodies and during the washing procedures. During incubation with the sample serum detachment of antigen was quite rapid and could result in competition between bound and free antigen for antibodies. The presence of antigen-antibody complexes in reaction mixtures could also lead to erroneous results in the antibody assay. With covalent coupling of antigen to solid phase (cyanogen bromide activated paper), batch-to-batch variation in the surface concentration of antigen was strikingly less than in the assay using plastics. Detachment of antigen from activated paper during the assay was less than with polystyrene or nylon. ELISA with activated paper as the solid phase thus seems suitable as a reference method for standardisation of the antigen phase in ELISA systems.

Preparations of purified immunoglobulins, light chains, and unrelated proteins (used as controls) were covalently linked to agarose beads and used to study the specificity of fluorescein isothiocyanate conjugates. The data demonstrate how these beads can be used to detect immunological and non-immunological reactivity in conjugates. Commercial conjugates, conjugates prepared in this laboratory, and fluorescein-labeled normal immunoglobulins demonstrated high reactivity with control beads unless chromatographed on diethylaminoethyl-cellulose to select for the proper fluorescein-protein ratio. Undesirable immunological reactivity could be demonstrated in commercial conjugates and was shown to be due to anti-light-chain antibody.

We report here the primary structure of an immunoglobulin heavy chain synthesized by ICR 16, a variant of the MPC 11 mouse myeloma cell line. The ICR 16 heavy chain is a gamma 2b-gamma 2a hybrid, consisting of the CH1 domain of gamma 2b and the hinge, CH2 and CH3 domains of gamma 2a subclasses. The genetic mechanism by which ICR 16 occurred may be recombination, based on homologies in both coding and intervening sequences in gamma 2b and gamma 2a constant region genes. Although the Fc fragment of ICR 16 is completely gamma 2a-like and has been shown to bind to gamma 2a Fc receptors on mouse macrophages, the intact H2L2 molecules is unable to do so. Such an observation underscores the crucial role that conformation may play in the ability of immunoglobulins to carry out biologic functions.

Structural studies were carried out on a monotypic immunoglobulin (Ig) isolated from a patient suffering from a colon tumor. Results indicated that the light (L) chain of this protein belonged to the VkappaII subgroup and was devoid of known Inv allotypic determinants, whereas the heavy (H) chain variable (V) region belonged to the VHIII subgroup and its constant (C) region was of the gamma1 subclass and was Gm (a+Z+). The amino acid sequence of a total of 106 residues has been determined for this molecule. An extra cysteine was present at the fourth hypervarible region of the heavy chain. Preliminary results indicated that the Fc fragment of this protein did not include the inter-heavy-chain disulfide bonds.

The chondroblastic component of a human osteosarcoma was established as a transplantable tumor in nude athymic mice for the first time. The collagen biosynthetic production of the excised tumor was studied in organ culture. The majority of the collagen secreted in the culture media was pro-Type II as identified by diethylaminoethyl cellulose (DEAE cellulose) chromatography, gel electrophoresis, and cyanogen bromide cleavage. This system should be useful for producing large quantities of human pro-Type II collagen and for studying neoplastic cell switching of collagen synthesis.

Pregnancy-associated plasma protein A (PAPP-A) has been obtained in an enriched state under mild conditions of purification involving cibacron blue dye-ligand chromatography, negative antibody-affinity chromatography and gel filtration. The product was a mixture of PAPP-A and alpha-2-macroglobulin (alpha 2M). The ability of these proteins to bind 125I-trypsin and 125I-plasmin was studied. In contrast to previous studies by others, there was no evidence that PAPP-A could bind either protease. It is pointed out that protease-inhibitory activity in samples of PAPP-A can be due to the presence of very small amounts of contaminating alpha 2M in the preparations of PAPP-A. In further experiments PAPP-A did not inhibit the complement-induced lysis of sensitized red cells. Finally, cyanogen bromide peptide mapping experiments failed to yield evidence of structural homologies between PAPP-A and alpha 2M.

Phenylalanine chloromethyl ketone covalently attached to porous glass beads was synthesized to serve as a solid-phase active site directed inhibitor of chymotrypsin-like proteolytic enzymes. The solid-phase reagent inhibited 20 nmol of bovine chymotrypsin per gram of glass and covalently bound 30 nmol of protein per gram of glass. Sepharose-bound lysine chloromethyl ketones were synthesized to serve as inhibitors of trypsin-like enzymes. Sepharose-MethionylLysyl chloromethyl ketone inactivated and bound about 6.8 nmol of enzyme per ml of settled gel. In a preliminary experiment, a cyanogen bromide cleavage of the methionine residues showed that it should be possible to release all peptides but the peptide containing the active-site histidine. The immobilized trypsin was also reduced, carboxymethylated and digested with chymotrypsin. The potential of the solid-phase approach is in the isolation of a specific serine proteinase and in the sequence determination of residues surrounding the active-site histidine.

A 3.3-kilobase DNA complementary to human microtubule-associated protein 2 (MAP2) was sequenced by the dideoxy method. The 3' end terminates at an internal EcoRI site before the polyA tail. Due to the arrangement of the cDNA insert in the lambda gt11 vector, the MAP2 fragment is not fused to beta-galactosidase when expressed. The Chou Fasman algorithm for the initial 58 amino acids from the first in-frame methionine predicts an alpha helix. Beyond this point, a series of turns is predicted until amino acid 160. The frequent presence of basic residues in proximity to serines or threonines is consistent with multiple phosphorylation sites. The minimum specificity determinant for Ca2+/calmodulin-dependent kinase is repeated 13 times. The sequence of a region containing a MAP2 epitope that is shared with the Alzheimer neurofibrillary tangle was determined by DNase treatment of the cDNA and antibody selecting the small resultant clones in a lambda gt11 sublibrary. Likewise, a MAP2 epitope that is not shared with the neurofibrillary tangle also has been located. Both epitopes are in the projection portion of the molecule. A bovine MAP2 cyanogen bromide fragment, which contains the epitope shared with the neurofibrillary tangle, is partially insoluble under aqueous conditions, probably due to the aggregation of oppositely charged residues. Thus, rapid cleavage of MAP2 to small peptides is probably necessary in vivo to prevent the aggregation of larger cleavage fragments.

The in vitro phenotype of bovine articular chondrocytes is described. Chondrocytes plated at high density in roller-bottle and dish cultures were maintained in vitro. The major matrix macromolecules, collagen and proteoglycan, synthesized by these cells were characterized during the course of the culture period. The chondrocytes synthesized mainly Type II collagen, which was found predominantly in the cell-associated matrix. The media contained a mixture of Type II and Type III collagens. Type I collagen was detectable in neither the medium nor the cell-associated matrix. The proteoglycan monomers found in media and cell-associated matrix had the same hydrodynamic sizes as monomers synthesized by cartilage slices or those extracted from adult articular cartilage. The majority of proteoglycans synthesized by the cells were found in high molecular weight aggregates which were readily recovered from the media and were extractable from cell-associated matrix with low ionic strength buffers. The results demonstrate the long-term in vitro phenotypic stability of the bovine articular chondrocytes. The advantages of the in vitro system as a model for studying the effects of external agents, such as drugs and vitamins, are discussed.

For coupling proteins to Sephacryl gels periodate oxidation of these gels was investigated as an alternative method to cyanogen bromide activation. Optimum conditions were studied with respect to periodate concentration, time of oxidation, pH and type of coupling buffer, concentration of protein, temperature and time of protein uptake, and protein leakage after coupling. The effects of sodium cyanoborohydride and ascorbic acid as reducing agents, and of manganese ions as a potential catalyst were investigated. Using the derived optimum conditions, stable solid-phased antibodies were produced in high yield and used to adsorb factor VIII from plasma. These gels were stable for many weeks, as was the intermediate oxidised gel. Reductive amination for coupling proteins to oxidised Sephacryl gels results in increased binding and lower leakage than is obtained with cyanogen bromide activated agarose.

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.

A peptide of unknown structure was found as a side product in a commercial preparation of the 1-34 fragment of bovine parathyroid hormone (PTH). CNBr cleavage and amino acid analysis showed that this peptide is the des-lys-13 form of 1-34 bovine PTH. The peptide thus represents a deletion mutant of PTH and structure-function studies are of interest. This peptide was a full agonist in the adenylyl cyclase bioassay for PTH, but its potency was about 5% of that found for the complete 1-34 peptide. Proton NMR studies showed that the pK values for the histidine residues in the des-lys-13 form were essentially identical to those of the intact peptide. However, pH-dependent changes in the chemical shifts for the tryptophan protons (residue 23) and several unidentified methyl group resonances were observed in the des-lys peptide. The latter are major shifts and probably represent ring-current effects; these were not seen in the intact 1-34 peptide. The results show that Lys-13 is important in the folding of the active domain of PTH, and are interpreted in the context of a previously published model for the folding of this hormone.

Lyophilized bovine, porcine, and human choroid plexuses contain .02-.09 U of antidiuretic activity per milligram. The antidiuretic factor in bovine choroid plexus was concentrated 100 times by extraction with acetic acid, fractional precipitation with acetone and ethyl ether, gel filtration, and paper chromatography. Resulting choroid plexus fraction IIgammaB2 was eluted from Sephadex G-25 in position corresponding to molecular weight between 750 and 3,500; its antidiuretic activity was destroyed by trypsin, performic acid, and thioglycollic acid, but was not affected by leucine aminopeptidase, carboxypeptidase A or B, or cyanogen bromide. HgammaB2 possesses antidiuretic, pressor, and oxytocic potencies (measured in anesthetized-hydrated rat, anesthetized rat, and isolated rat uterus, respectively) of 1.9, 0.5, and 0.1 U/mg, respectively.

Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin.

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.

Pyridoxal phosphate-dependent histidine decarboxylase from Morganella morganii AM-15 was inactivated by (S)-alpha-fluoromethylhistidine by a pseudo first-order reaction, with KI and k inact values of 0.1 mM and 32.2 min-1, respectively, and was most efficient at pH 6.5-7.0. Both L-histidine and the competitive inhibitor, L-histidine methyl ester, protected against inactivation. The apoenzyme was not inactivated. These findings indicate that inhibition is a mechanism-based process. Under optimal conditions a single molecule of alpha-fluoromethylhistidine inactivates one enzyme subunit, indicating that no escaping side reaction occurs during the inactivation process. The bound inactivator is not released by dialysis of the native protein but is released upon denaturation by heat or urea. This released product was not fully characterized, but it contains the tritium of ring-labeled alpha-fluoromethyl-[3H]histidine, exhibits the spectral properties of a 3-hydroxypyridine derivative, and does not yield any amino acids on hydrolysis. The label was much more stable following borohydride reduction of the inactivated protein, and a tryptic peptide containing the modified residue was isolated. Sequencing of this peptide and the corresponding peptide from the native enzyme revealed that the inactivator binds to a serine residue of the holoenzyme. Two P-pyridoxyl peptides from tryptic or CNBr digests of the NaBH4-reduced enzyme were also isolated. Sequence and compositional data obtained with these peptides showed that the serine residue to which the inhibitor binds is not near the lysine residue that binds pyridoxal-P in the primary sequence of the protein, although the two residues must be near one another in the three-dimensional structure to account for these results. A speculative mechanism for inactivation, consistent with the experimental findings, is presented.

This paper presents definitive results which establishes a direct gene-protein product relationship between the pgsA gene and the phosphatidylglycerophosphate synthase of Escherichia coli. The predicted protein sequence derived from the determined DNA sequence of pgsA is in close agreement with the amino acid composition and partially determined amino acid sequence of the purified enzyme. The purified synthase has the same apparent molecular mass as the gene product made by a plasmid-directed transcription-translation system. The plasmid-borne copy of the pgsA gene is also capable of expressing enzymatically active synthase in vitro. The DNA sequence analysis has established the exact linear relationship between the uvrC, pgsA, and glyW loci and revealed that these three genes are transcribed in the same direction. The terminal coding regions of these three genes also share common sequences with transcriptional regulatory elements for the adjacent genes.

Stable azide complex of cyanogen bromide-modified met-myoglobin (metMb) was prepared and crystallized. The principal values and eigen vectors of g-tensor were determined by single-crystal EPR spectroscopy at 77 K: gxx = 1.50, gyy = 2.32, and gzz = 2.91. These g values were similar to those of tetrazole derivative rather than azide derivative of native metMbs, suggesting that tetrazole derivative might be formed from N-cyanoimidazole of distal histidyl residue via nucleophilic attack of azide ion by 1,3-dipolar cycloaddition reaction. The orientation of the maximal g value (gzz) of the novel product was found to deviate about 13 degrees from the heme normal of native aquometMb. Thus, the orientation of the heme plane might be altered in passing from native metMb to cyanogen bromide-mediated metmyoglobin. The present EPR results demonstrated that the modification of the histidyl residue at the heme distal side causes the changes in the stereochemical and electronic natures of the ligand binding to the heme.

Fifteen aminoacyl-transfer ribonucleic acids (tRNA's) from vegetative cells (trophozoites) and mature cysts of Acanthamoeba castellanii were compared by reversed-phase 5 chromatography. Little or no differences were detected in reversed-phase 5 chromatography elution profiles of alanyl-, arginyl-, isoleucyl-, phenylalanyl-, prolyl-, seryl-, threonyl-, tryptophanyl- and valyl-tRNA's. Significant differences in the relative proportions of isoaccepting species of leucyl-, lysyl-, methionyl-, aspartyl-, histidyl-, and tyrosyl-tRNA's were observed. Based upon the criterion of cyanogen bromide reactivity with the modified nucleoside queuosine, the content of queuosine in aspartyl-tRNA of A, castellanii is significantly greater in mature cysts than in trophozoites. The similarity of change in reversed-phase 5 chromatography elution profiles of aspartyl-, histidyl-, and tyrosyl-tRNA suggests that a common mechanism is responsible for alterations in the chromatographic patterns.

Vasoactive intestinal polypeptide (VIP) was labeled with sodium [125I]iodide using the chloramine-T method and subsequently purified by reverse-phase high performance liquid chromatography. Three main 125I-labeled peaks designated A, B, and C resulted from the radioiodination and purification procedures. They were characterized by electrophoresis of tryptic fragments; Edman degradation (for Peaks A and C); enzymatic digestion to amino acids by leucine aminopeptidase, carboxypeptidase Y and Pronase; and treatment with cyanogen bromide. Peak A corresponds to VIP monoiodinated on Tyr10 and with the Met17 residue oxidized to methionine sulfoxide. This [mono[125I]iodo-Tyr10,MetO17]VIP displays the following characteristics. 1) It constitutes quantitatively the major product of the iodination procedure (62.5%); 2) it is well resolved from other labeled and unlabeled products; 3) it is stable (2 months at -20 degrees C); 4) it possesses a high specific activity (2050 Ci/mmol); 5) it maintains the biological activity of native VIP; and 6) it binds to antibody and membrane recognition sites in a specific, saturable, and reversible manner. Reduction of [mono[125I]iodo-Tyr10, Met-O17]VIP to [mono[125I]iodo-Tyr10]VIP does not improve the performance of the tracer in a radioimmunoassay. The method described in this article is simple and rapid and yields a molecular form of 125I-labeled VIP that has been fully characterized and is suitable for use in biological studies.

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.

The occasional cleavage of the Pseudomonas cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) molecule into two well-defined fragments during the preparation of the enzyme is shown to be identical to that caused by elastase isolated from the culture solution of Pseudomonas aeruginosa. A cyanogen bromide fragmentation of proteolytically cleaved and of intact enzyme shows the cleaved peptide bond to be situated in cyanogen bromide fragment II. The amino-acid sequence of this fragment was established by sequencing peptides obtained with trypsin, thermolysin, chymotrypsin and o-iodosobenzoate. It is concluded from the sequence homology that the polypeptide chain of Pseudomonas peroxidase is wrapped around the high-potential heme in a similar manner as in high-potential cytochromes c in general. The specific proteolytic cleavage occurs at a Ser-Val (Leu-Pro) region which is assumed to be the site of attachment between enzyme and membrane. The cleavage of the Ser-Val bond renders the peroxidase molecule enzymatically inactive by impeding the conformational changes essential for the function of the native enzyme.

A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.

The proliferative and helper T cell repertoires were compared in the CBA/J mouse for the response to the large protein antigen, tetrameric beta-galactosidase (GZ = 1021 a.a/monomer). The systems assessed the ability of cyanogen bromide (CB) peptides of GZ to: 1) prime for a T cell proliferative response to GZ; or 2) generate T cell help, measured by the production of anti-FITC PFC in the in vitro response to GZ-FITC. Priming for in vitro proliferation was attempted with 11 CB peptides comprising 70% of the GZ molecule. Strong priming was found with five peptides and intermediate priming was found with four other peptides; two peptides were without effect (CB-20 = a.a 767-862, and CB-4 = a.a. 188-202). Despite this indication of generally dispersed recognition of GZ epitopes, only two CB peptides, CB-2 (a.a. 3-92) and CB-10 (a.a. 378-418) were able to induce a T helper cell response. The surprising dearth of helper T cell-inducing epitopes may be peculiar to the limited fluorescein (FITC) substitution on GZ-FITC (17-25 FITC residues per tetramer) or it may reflect the constraints involved in T cell recognition required for T-B collaboration. Also considered was the possibility that the helper T cell repertoire might be distinct from the proliferative repertoire, the latter reflecting DNA synthesis and recruitment by other functional T cell subpopulations.

Conditions for the release of streptococcal type 12 M protein from whole cells by cyanogen bromide are described; they demonstrated that methionine is not essential to the structural arrangements which account for some of its immunological and biological properties. The released M protein was separated from other proteins by column chromatography with hydroxylapatite. The type-specific molecules which reacted with precipitating antibodies were found only in the 0.3 M eluate, formed zones with mobilities less than 12% of that of the dye front on electrophoresis in the standard acrylamide disc gel system, formed at least four bands in sodium dodecyl sulfate-acrylamide disc gels with molecular weights ranging from 12,000 to 23,000, and stimulated the formation of opsonic antibodies in rabbits. Cyanogen bromide provides a highly specific method for the release of M proteins which should prove particularly useful in analyses of structural-functional relationships among different M proteins.

Biochemical analysis of the H-2K-gene product from the MHC mutant strain bml and from the C57BL/6 parent strain has been carried out in order to characterize the structural differences between parent and mutant K-gene products. Based on comparative tryptic peptide mapping of the cyanogen bromide fragments from these glycoproteins, two peptide differences were localized to the CN-Ia fragment. Partial amino-acid sequence analysis revealed two alterations in the primary structure of Kbml involving substitutions of tyrosine for arginine at position 155, and tyrosine for leucine at position 156. Both of these amino-acid replacements require a minimum of two nucleotide base changes at the nucleic acid level. These changes were the only alterations noted differentiating the Kbml and Kb glycoproteins. However, because our techniques allow us to analyze only 75 to 80 percent of the extra cellular portion of H-2Kb, it is possible there are other undetected changes. Nonetheless, the biochemical data are consistent with the hypothesis that the structural alterations noted in the Kbml mutant glycoprotein are directly related to the observed immunological specificity relative to the parent Kb molecule. Peptide comparisons of the Kb molecules of two C57BL/6 sublines and of the H-2b lymphoblastoid cell line, EL-4, disclosed no difference.

A rat brain S100-binding protein, R40,000, has been isolated, characterized, and identified as fructose-1,6-bisphosphate aldolase. R40,000 was purified by ammonium sulfate precipitation, hydroxylapatite chromatography, dye-binding chromatography, and electroelution from sodium dodecyl sulfate-polyacrylamide gels. Microsequence analysis of a fragment of R40,000 revealed a 15-residue amino acid sequence which shows a high degree of homology to the amino acid sequence of fructose-1,6-bisphosphate aldolase from rabbit muscle and rat liver. Further characterization demonstrated that R40,000 has an amino acid composition, subunit molecular weight, and cyanogen bromide map similar to aldolase. In addition, purified aldolase interacts with S100 alpha and S100 beta by gel overlay, and aldolase enzyme activity is stimulated 2-fold in vitro by S100 alpha and S100 beta. S100 interacts predominantly with the C or brain-specific form of the enzyme in gels and stimulates the activity of the C-enriched form of the enzyme in a calcium-dependent manner. Altogether, these data suggest that fructose-1,6-bisphosphate aldolase may be an intracellular target of S100 action in brain.

The complex between lactase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) and phlorizin hydrolase (glycosyl-N-acylsphingosine glycohydrolase, EC 3.2.1.62) has been purified from the proximal and distal regions of the small intestine of suckling rats. The two enzymes behaved differently on DEAE-cellulose ion-exchange chromatography and during electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS), but they have very similar cyanoge bromide cleavage patterns. Kinetic studies on the proximal and distal enzymes showed the same pH optimum of 6.0 and the same heat stability at 45 degrees C, but a small difference in Km. Treatment of both enzymes with fucosidase, mannosidase or N-acetylhexosaminidase did not affect enzymic activity or electrophoretic mobility. Neuraminidase digestion abolished the electrophoretic differences and gave two active enzymes with similar isoelectric points.

Monoclonal antibodies against an endogenous beta-galactoside-binding lectin (monomer molecular weight 14,000, 14K lectin) of chick embryo were prepared and characterized. The inhibitory activities against hemagglutination, antigenic determinants and binding specificities were examined. Monoclonal antibody S1A4-5 strongly inhibited the hemagglutination activity of this lectin. This antibody did not bind to any cyanogen bromide (BrCN) fragment of the lectin. Another monoclonal antibody, S1A4-3, bound to one of the BrCN fragments (residues 34-66). However, this antibody inhibited hemagglutination only weakly. The bindings to isolectins of beta-galactoside-binding lectin, namely 14K lectin (monomer molecular weight 16,000) and a third species which is assumed to be a hybrid molecule consisting of 14K and 16K lectin subunits, were examined. The antibody SIA4-5 bound to 14K lectin but not to 16K lectin. In the case of the third species, intermediate binding was observed.

The complete amino acid sequence of pilin from gonococcal strain MS11 and the sequence of constant and variable regions from strain R10 pilin have been determined in order to elucidate the structural basis for adherence function, antigenic diversity, and polymeric structure. The MS11 pilin sequence consists of 159 amino acids in a single polypeptide chain with two cysteines in disulfide linkage and serine-bonded phosphate residues. TC-2 (31-111), a soluble monomeric pilus peptide prepared by arginine-specific digestion, bound human endocervical, but not buccal or HeLa cells and therefore is postulated to encompass the receptor binding domain. Variable regions of CNBr-3 appear to confer antigenic diversity and comprise segments in which changes in the position of charged residues occur in hydrophilic, beta-turns. Residues 2-21 and 202-221 of gonococcal pilins and lower eucaryotic actins, respectively, exhibit 50% homology. When these residues are arranged at intervals of 100 degrees of arc on "helical wheels," the identical amino acids comprise a hydrophobic face on one side of the helix. This observation, the hydrophobic character of this region and the tendency for TC-1 (residues 1-30) to aggregate in water, suggest that this stretch interacts with other subunits to stabilize polymeric structure.

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).

Monoamine oxidases A and B (amino: oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4) have been identified in the outer membranes of rat liver mitochondria by their covalent reaction with the inhibitor, [3H]pargyline. On analysis by polyacrylamide gel electrophoresis under denaturing conditions. Monoamine oxidase A was found to migrate more slowly that monoamine oxidase B. Proteins which correspond to monoamine oxidases A and B (as identified by the electrophoretic distribution of covalently bound [3H]pargyline) were excised from the gels. Subsequent analysis showed that both monoamine oxidase A and monoamine B had been highly purified by this procedure. Electrophoretic analysis of the peptides produced by limited proteolysis with bovine trypsin, alpha-chymotrypsin, Staphylococcus aureus V8 proteinase and cyanogen bromide indicate that monoamine oxidases A and B have different amino acid sequences.

We report a simple method to obtain internal amino acid sequences from larger proteins electroblotted to polyvinyldifluoride membranes. To demonstrate this method, immunoglobulin heavy and light chains are separated by gel electrophoresis and electroblotted to the membrane. The separated chains, immobilized to the membrane, are cleaved in situ by cyanogen bromide and the resulting fragments are subsequently eluted from the membrane. The fragments are separated by gel electrophoresis, electroblotted and subjected to gas-phase microsequence analysis.

The myeloma proteins binding inulin afford a unique opportunity to study the V region patterns of variation because of the similarity in the VL as well as the VH regions. The diversity patterns in both the VL and VH regions suggest these proteins are encoded by multiple, very similar V gene segments or that somatic mutation may repeatedly generate identical variants. Because of the close similarity in the V domains of these proteins and the extensive idiotypic analyses that have been carried out previously, several interesting conclusions can be drawn about the nature of idiotypic determinants. First, a single amino acid residue may be involved in determining multiple idiotypic determinants. Second, hapten-inhibitable idiotypes may depend on residues within and outside the hypervariable regions. Third, idiotypic similarity does not always predict a corresponding sequence similarity.

Type V collagen from FBJ virus-induced osteosarcoma of mice has a high content of saccharide as has been noted for type V collagens from different sources. In the present study, this collagen was found to contain significant amounts of mannose and hexosamine. Three alpha chains of this collagen were electrophoretically separated and cleaved by cyanogen bromide. The cyanogen bromide peptides, following their separation by SDS-polyacrylamide gel electrophoresis, were transferred onto nitrocellulose paper and stained with concanavalin A. Several bands derived only from alpha 3(V) stained positively, but this was inhibited by the presence of alpha-methylmannoside. Thus, at least one of these three chains appears to have an asparagine-linked oligosaccharide.

In previous studies, the single selenonucleoside component of a selenium-containing tRNAGlu isolated from Clostridium sticklandii has been shown to be 5-methyl-aminomethyl-2-selenouridine. Here, we show that this selenonucleoside is most likely located at the "wobble" position of the anticodon of the clostridial seleno-tRNAGlu. Nuclease T1 digestion of this seleno-tRNAGlu generated one major selenium-containing oligonucleotide (25 bases long). The selenium-containing residue within this oligonucleotide was located by sequence analysis of the oligonucleotide before and after removal of selenium by treatment with cyanogen bromide. The sequence of this oligonucleotide, A-A-C-C-G-C-C-C-U-U+-U-C-A+C-G-G-C-G-G-U-A-A-C-A-G, is homologous to that of the Escherichia coli tRNAGlu2 from residues 27 to 50, including the anticodon region and the variable loop, except that the E. coli tRNA has 5-methylaminomethyl-2-thiouridine instead of the selenonucleoside.

We have cloned and sequenced a full-length cDNA coding for human uroporphyrinogen decarboxylase. The deduced 367-amino acid sequence is consistent with the molecular weight, the partial amino acid sequence of cyanogen bromide peptides, and the total amino acid composition of the purified enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome, and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. We have also demonstrated that the level of uroporphyrinogen decarboxylase mRNA is markedly increased in tissues or cell lines of erythroid origin and that this is due to a tissue-specific transcriptional activation of the uroporphyrinogen decarboxylase gene.

Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.

The sequence of the 240 amino acids and the position of the five S-S bridges of subunit III of the bovine pancreatic 6 S procarboxypeptidase A complex have been determined thus confirming its phylogenetic filiation with the pancreatic serine endopeptidase group. The subunit contains at equivalent positions all the elements of the catalytic site of these enzymes. The elements of a binding pocket very similar to that of porcine elastase I are also present in the protein thus accounting for its zymogen-like activity. The most obvious difference is the absence in the subunit of the two strongly hydrophobic amino acids (16 and 17 in the chymotrypsinogen numbering), which are known to participate in the stabilization of a fully functional binding pocket in active endopeptidases. Four of the five disulfide bridges of subunit III are homologous with those common to all pancreatic endopeptidases. In contrast the fifth bridge forms a very small loop of only four amino acids, which is not encountered in active endopeptidases. Other potentially lethal modifications in the structure of the subunit are not excluded.

When primary corneal endothelial cells were grown in polymorphonuclear leukocyte (PMN)-conditioned medium, a minor population of cells acquired fibroblastic morphology. Such modulated endothelial cells supported by PMN-conditioned medium grew much faster than the major nonresponding polygonal endothelial cell. Upon serial passages, the modulated endothelial cells became the dominant cell type and eventually formed a homogeneous fibroblastic culture. At the same time, phenotypic changes of collagen were observed. The primary endothelial cells grown in PMN-conditioned medium, consisting of responding elongated cells and nonresponding polygonal endothelial cells, produced predominantly type IV collagen with type III collagen as a minor component. As cells were subcultured and fibroblastic cells became dominant, type IV collagen synthesis was dramatically decreased and type I collagen synthesis was increased in parallel. When they reached the fully modulated stage, the cultures synthesized types I and III collagen, with type I accounting for 75-85% of the total. Type I collagen synthesized by the fibroblastic endothelial cells shared common characteristics with known type I collagen, such as migration behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CNBr peptide profiles, and immunologic identity. Thus, PMNs apparently contribute to the modulation of corneal endothelial cells, causing them to acquire characteristics of fibroblasts, cell multilayering, and deposition of interstitial extracellular matrix composed predominantly of interstitial type I collagen.

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.

Multiple fibronectin mRNAs arise by alternative splicing of the primary transcript of a single gene. We describe analyses of the contribution of this alternative splicing to fibronectin subunit heterogeneity in three different cell types using antisera directed against specific segments of fibronectin. beta-galactosidase-fibronectin fusion proteins produced with the lambda gt11 bacterial expression vector were used as immunogens. One region of alternative splicing accounts for differences in subunit size, while a second contributes to differences between the fibronectins present in blood plasma and in fibroblastic cells. We also show, however, that these two regions of alternative splicing do not account for all detectable subunits. We have also used these segment-specific antisera to show that blood platelets contain a spectrum of fibronectin subunits distinct from that found in blood plasma.

The amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor) was determined by automated Edman degradation using the peptides produced by chemical and enzymatic cleavages of intact factor XII and alpha-factor XIIa. Combining this sequence with the previously determined sequence of beta-factor XIIa (Fujikawa, K., and McMullen, B. A. (1983) J. Biol. Chem. 258, 10924-10933), the complete amino acid sequence of human factor XII has been established. The heavy chain of alpha-factor XIIa is composed of 353 amino acid residues containing one Asn-linked and six probable O-linked carbohydrate chains. The heavy chain of alpha-factor XIIa appears to contain four different domains including a "kringle," a "growth factor" domain, and the "type I" and "type II" domains of fibronectin. The domain organization of factor XII is analogous to those of several fibrinolytic proteins, including tissue plasminogen activator and urokinase, suggesting that factor XII belongs to the same protease subfamily as these two proteins.