Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum.

1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively.     

Antithrombin III and its interaction with heparin. Comparison of the human, bovine, and porcine proteins by 1H NMR spectroscopy

1H NMR has been used to characterize and compare the structures of antithrombin III from human, bovine, and porcine plasma as well as to investigate the interactions of each of these proteins with heparin fragments of defined length. The amino acid compositions of the three proteins are very similar, which is reflected in the gross features of their 1H NMR spectra. In addition, aromatic and methyl proton resonances in upfield-shifted positions appear to be common to all three proteins and suggest similar tertiary structures. Human antithrombin III has five histidine residues, bovine has six, and porcine has five. The C(2) proton from each of these residues gives a narrow resonance and titrates with pH; the pKa's are in the range 5.15-7.25. It is concluded that all histidines in each protein are surface residues with considerable independent mobility. The carbohydrate chains in each protein also give sharp resonances consistent with a surface location and motional flexibility. The 1H spectra are sensitive to heparin binding. Although heparin resonances obscure protein resonances in the region 3.2-6.0 ppm, difference spectra between antithrombin III with and without heparin show clear perturbation of a small number of aromatic and aliphatic protein protons. These resonances include those of histidine C(2) and C(4) protons, of 10-20 other aromatic protons, of a methyl group, and also of protons with chemical shifts similar to those of lysine and/or arginine side chains. For human antithrombin III, it was shown that heparin fragments 8, 10, and 16 sugar residues in length result in almost identical perturbations to the protein. In contrast, tetrasaccharide results in fewer perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the domain structure of antithrombin III. Tentative localization of the heparin binding region using 1H NMR spectroscopy

The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by 1H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053] at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to be homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.     

Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.     

Renal gamma-glutamyl transpeptidases: structural and immunological studies

Mammalian kidney gamma-glutamyl transpeptidases are compared with respect to subunit size, amino-terminal sequences of the two subunits, immunological, and some catalytic properties. The species-related variation in the apparent molecular weight of the subunits has been shown to be primarily due to the extent and nature of protein glycosylation. Using antibodies raised against the native enzymes and isolated sodium dodecyl sulfate-treated subunits, it is shown that the transpeptidases share some antigenic determinants. Some of these determinants in the highly glycosylated transpeptidase subunits can be detected by the antibodies only upon deglycosylation of the subunits. The amino-terminal sequences of the subunits exhibit considerable homology, in agreement with the immunological data. Thus, there are two segments of identity (3 and 5 residues in length, respectively) in the first 17 amino-terminal residues of the heavy subunits of rat, bovine, dog, and human kidney transpeptidases (papain-solubilized). Of particular interest is the finding of 91 to 96% identity in the first 23 amino-terminal residues of the small subunit of these transpeptidases. The small subunit contains the gamma-glutamyl binding site of the enzyme. There are three segments of identity (7, 6, and 8 residues in length, respectively) in the first 23 residues, each separated by either a Ser or an Ala residue. The first 7 amino-terminal residues of the small subunit in all four species are identical, indicating a high degree of specificity in the proteolytic processing of the common, single-chain precursor of the two subunits. Differences noted between transpeptidases in their relative acceptor specificity and in their susceptibility to inactivation by the glutamine antagonist, AT-125 (acivicin), must reflect subtle structural differences in their active center domains.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

Probing the heparin-binding domain of human antithrombin III with V8 protease

From structural analysis on genetically abnormal and chemically modified human antithrombin III [Koide, T., Odani, S., Takahashi, K., Ono, T. and Sakuragawa, N. (1984) Proc. Natl Acad. Sci. USA 81, 289-293; Chang, J.-Y. and Tran, T. H., (1986) J. Biol. Chem. 261, 1174-1176; Blackburn, M. N., Smith, R. L., Carson, J. and Sibley, C. C. (1984) J. Biol. Chem. 259, 939-941], the heparin-binding site of antithrombin III has been suggested to be in the region of Pro-41, Arg-47 and Trp-49. In this study the heparin-binding site was probed by preferential cleavage of V8 protease on heparin-treated and non-treated native antithrombin III. The study has been based on the presumption that the heparin-binding site of antithrombin III is situated at exposed surface domain and may be preferentially attacked during limited proteolytic digestion. Partially digested antithrombin III samples were monitored by quantitative amino-terminal analysis and amino acid sequencing to identify the preferential cleavage sites. 1-h-digested antithrombin III was separated on HPLC and peptide fragments were isolated and characterized both qualitatively and quantitatively. The results reveal that Glu-Gly (residues 34-35), Glu-Ala (residues 42-43) and Glu-Leu (residues 50-51) are three preferential cleavage sites for V8 protease and their cleavage, especially the Glu-Ala and the Glu-Leu sites, was drastically inhibited when antithrombin III was preincubated with heparin. Both high-affinity and low-affinity antithrombin-III-binding heparins were shown to inhibit the V8 protease digestion of native antithrombin III, but the high-affinity sample exhibited a higher inhibition activity than the low-affinity heparin. These findings (a) imply that the segment containing residues 34-51 is among the most exposed region of native antithrombin III and (b) support the previous conclusions that this region may play a pivotal role in the heparin binding.     

Properties of thrombin- and elastase-modified human antithrombin III

Proteolytically modified forms of human antithrombin III have been prepared by reaction of native antithrombin with thrombin, human neutrophil elastase, or porcine pancreatic elastase. These forms have two chains disulfide linked and are of the same molecular weight as native antithrombin III. 1H NMR spectroscopy has been used to characterize these proteins and to compare them to one another and to native antithrombin III. The three modified proteins have very similar NMR spectra and histidine residues with identical pH titration parameters, and they undergo the same spectral changes upon binding heparin. They differ from native antithrombin III in all of these respects. In addition, the proteins are much more stable than native antithrombin III. The three modified proteins behave identically as a function of temperature; at 372 K, 44 K above the unfolding temperature for native antithrombin III, the proteins are still folded and possess approximately 70 unexchanged amide protons even after several hours. The unfolding of the heparin binding domain at low concentrations of deuteriated guanidine hydrochloride seen in native thrombin III is absent in the modified forms. It is concluded that the thrombin- and elastase-modified forms of antithrombin have identical structures when allowance is made for the slightly different sites of cleavage by the two types of elastase and by thrombin. This structure is very different from that of native antithrombin III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma.

Protein S is a vitamin K dependent protein of unknown function, which is present in mammalian plasma. It was isolated from bovine plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, and column chromatography on DEAE-Sephadex, heparin-agarose, and polyhomoarginine-Sepharose. Bovine Protein S (Mr 64,200) is a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Thr-Leu-Leu-. It contains 7.0% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. Human Protein S (Mr 69,000) is also a single-chain glycoprotein with an amino-terminal sequence of Ala-Asn-Ser-Leu-Leu-. It contains 7.8% carbohydrate and 10 residues of gamma-carboxyglutamic acid per mol of protein. These results indicate that Protein S from bovine or human plasma shows many similarities to the other vitamin K dependent proteins present in plasma.     

The heparin binding site of human antithrombin III. Selective chemical modification at Lys114, Lys125, and Lys287 impairs its heparin cofactor activity

Heparin binds to human antithrombin III and accelerates its inhibitory activity in the blood coagulation system. Previous reports (Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505; Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938) have shown that selective chemical modification of a limited number of lysine residues in antithrombin III causes drastic loss of its heparin cofactor activity. We have performed chemical modification of antithrombin III with trinitrobenzene sulfonic acid in order to determine the location of these lysine residues. When antithrombin III was treated with 100 M excess of trinitrobenzene sulfonic acid for 10 min, about 3.2 mol of amino group per mol of antithrombin III were modified. The heparin cofactor activity dropped to about 25%, whereas the progressive inhibitory activity (in the absence of heparin) remained essentially intact (about 95%). The modified amino groups were identified to be Lys114 (75%), Lys125 (94%), and Lys287 (96%). These results were obtained by comparing and analyzing the cyanogen bromide fragments derived from native antithrombin III and the 10-min modified antithrombin III. When antithrombin III was pretreated with heparin, followed by trinitrobenzene sulfonic acid modification, the extent of modification at Lys114 and Lys125 decreased from 75% and 94% to 20% and 40%, respectively, whereas the modification at Lys287 remained nearly quantitative (greater than 95%). Based on these results, we conclude that Lys114 and Lys125 are essential for the heparin cofactor activity of human antithrombin III.     

Molecular weight analysis of antithrombin III-heparin and antithrombin III-thrombin-heparin complexes

The molecular interactions between components of the heparin-catalyzed antithrombin III/thrombin reaction were investigated by light scattering. When heparin was added to antithrombin III, the molecular weight increased to a maximum and then decreased to that of a 1:1 (antithrombin III X heparin) complex. The initial molecular weights at low heparin to antithrombin III ratios were consistent with the formation of a 2:1 (antithrombin III X heparin) complex in which only one antithrombin III molecule had undergone the conformational change measured by protein fluorescence enhancement. The peak molecular weight never reached that of a complete 2:1 complex. This behavior was observed for bovine and human antithrombin III in the presence of both unfractionated heparin and high molecular weight-high affinity heparin. Pentosane polysulfate also caused some multiple associations. Bovine antithrombin III and thrombin formed a 1:1 complex that underwent further aggregation within minutes, while the human proteins did not aggregate on this time scale after forming the 1:1 complex. In the presence of stoichiometric amounts of heparin, the bovine proteins formed an initial complex of Mr = 230,000 (corresponding to a dimer of heparin-antithrombin III-thrombin) which underwent further aggregation. The human proteins, however, formed a 1:1 (antithrombin III X thrombin) initial complex in the presence of heparin, followed by aggregation. These interactions of thrombin and antithrombin with heparin suggest complex interactions that could relate to heparin function.     

Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity

Antithrombin III Basel is a hereditary abnormal antithrombin with normal progressive inhibition activity (normal reactive site) and reduced heparin cofactor activity (impaired heparin binding site). Structures of antithrombin III Basel and normal antithrombin III isolated from the same patient were compared by peptide mapping using the dimethylaminoazobenzene isothiocyanate precolumn derivatization technique. Of the approximately 50 tryptic peptides of normal and abnormal antithrombin III, one peptide comprising residues 40-46 had a different retention time in reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide from antithrombin III Basel had a single substitution of Pro (normal) by Leu (abnormal) at position 41. This substitution is close to an Arg (residue 47) and a Trp (residue 49) which have previously been shown to be critical for heparin binding by antithrombin III. Although additional amino acid substitutions in antithrombin III Basel cannot be ruled out, this Pro-Leu replacement could cause a conformational change by increasing both the helical structure and the hydrophobicity around residue 41. These data suggest that: (i) the heparin binding site of antithrombin III encompasses the region containing residues 41, 47, and 49; and (ii) the impaired heparin cofactor activity of antithrombin III Basel is likely due to a conformational change of the heparin binding site induced by the Pro-Leu substitution at position 41.     

Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.     

Measurement of the affinities of heparins, naturally occurring glycosaminoglycans, and other sulfated polymers for antithrombin III and thrombin

Heparin, other glycosaminoglycans, and synthetic sulfated polymers have antithrombotic and anticoagulant activities, which may be mediated through a range of interactions with different proteins. A simple, quantitative method has been developed for assessing the affinity of interaction between sulfated polymers and proteins in the liquid phase. This has been used to compare the binding of a range of glycosaminoglycans and other sulfated polymers to antithrombin III and thrombin, a major inhibitor of and a central protease in the coagulation system, respectively. The results are consistent with the binding of naturally occurring glycosaminoglycans to antithrombin III solely through the well-defined antithrombin III-binding pentasaccharide found in heparin, the apparent affinity of a preparation depending upon its content of this pentasaccharide. Highly sulfated synthetic polymers will, however, bind antithrombin III by a second mechanism. The affinity of heparin for thrombin decreased with decreasing molecular weight. However, results obtained with heparan sulfate preparations did not indicate any clear relationship between either molecular weight or sulfate content and thrombin binding, but suggested that there may be an oligosaccharide sequence containing N-sulfate residues which confers high affinity for thrombin. In addition, some of the synthetic sulfated polymers bound thrombin with very high affinity.     

Molecular cloning of the E1 beta subunit of the rat branched chain alpha-ketoacid dehydrogenase.

We determined the cDNA sequence of the mRNA for antithrombin III (AT III) from sheep liver. It encodes a protein of 465 amino acids, including a signal peptide of 32 amino acids. The amino acid sequence of the mature protein shows a sequence identity of 89.1%, 95.6% and 85.0% to the human, bovine and rabbit equivalents, respectively. Cysteine residues involved in disulfide bonds as well as potential glycosylation sites are conserved between the four species. In contrast, the amino acid sequence of the signal peptide shows a smaller identity, i.e., 68.7% and 56.3% compared to the human and rabbit preprotein, respectively.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Comparison of the kinetic behavior of human and bovine proteins in the heparin-catalyzed antithrombin III/thrombin reaction

Significant differences between saturation kinetic properties of heparin-stimulated reactions between thrombin and antithrombin III from human and bovine species were observed. In both systems, the apparent Km for antithrombin III was higher than the KD for antithrombin III-heparin interaction, monitored by intrinsic protein fluorescence change. The Km for thrombin and kcat were much higher for proteins of the human species than the bovine species. The apparent Km for one human protein was dependent on the concentration of the other human protein, indicating interaction of the binding events. The reaction product formed from the bovine proteins was a potent inhibitor of the reaction but the product from the human proteins was a poor inhibitor. The major differences between the two species appeared to be related to interaction of thrombin or thrombin derivatives with heparin or heparin-antithrombin III complexes.     

Monoclonal antibodies against antithrombin III. Identification of their epitopes and effects on antithrombin III activities

Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.     

Isolation of two molecular weight variants of the mouse growth hormone receptor

GH receptors (GHRs) have been shown by affinity cross-linking to be present in late pregnant mouse liver microsomes in three forms with cross-linked mol wts of 125,000, 62,000, and 56,000. The two lower mol wt forms of the receptor were partially purified by bovine GH-affinity chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate-solubilized extracts of late pregnant mouse hepatic microsomes. The GHRs were identified from the partially purified receptor preparation and isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated GHRs had mol wts of 40,700 and 37,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Enzymatic cleavage of N-linked glycosylation from the isolated GHRs reduced their apparent mol wts to 33,600 and 30,900, respectively. Sixteen of the amino-terminal 17 amino acid residues of the two isolated receptors were sequenced and determined to be identical. One amino acid residue in each of the proteins, at position 14, could not be identified. Rabbit polyclonal antiserum was produced against the isolated GHRs. The resulting antiserum precipitated the isolated 40,700 and 37,500 mol wt proteins as well as cross-linked mouse GHRs (including the high mol wt form of the receptor). However, the antiserum did not inhibit the binding of mouse GH to either membrane bound or 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate-solubilized GHRs.     

Some sulfhydryl properties and primary structure of human erythrocyte superoxide dismutase

Human Cu-Zn superoxide dismutase prepared by different methods shows varying properties relevant to its sulfhydryl chemistry. A cysteine residue not found in the analogous bovine enzyme appears to be responsible for its unusual lability. Alkylation of this cysteine results in a marked increase in stability, and this form of the protein may be readily crystallized. The primary structure of the 153 amino acid residues found in the human protein has been determined, and 82% of the residues are identical with those of the bovine enzyme. A significant variation is seen in the portion of those proteins comprising residues 17-36, with eleven changes being noted.