Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure

Human alpha 2-macroglobulin (alpha 2M) half-molecules were prepared by limited reduction and alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule Mr approximately 180000 subunits into Mr approximately 90000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with alpha 2M whole molecules. The plasmin and trypsin binding capacities of the two forms of alpha 2M were compared by using radioiodinated proteases. alpha 2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced alpha 2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the two protease binding sites are adjacent in native alpha 2M. alpha 2M half-molecule-plasmin complexes reassociated less readily than half-molecule-trypsin complexes, supporting this interpretation. The frequency of covalent bond formation between plasmin and alpha 2M was considerably higher than that previously observed with other proteases. Approximately 80-90% of the plasmin that reacted with alpha 2M whole molecules or half-molecules became covalently bound. The reactivities of purified alpha 2M-plasmin complexes were compared with small and large substrates. Equivalent kcat/Km values were determined at 22 degrees C for the hydrolysis of H-D-Val-Leu-Lys-p-nitroanilide dihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s-1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s-1 determined for free plasmin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a "half-molecule" alpha 2-macroglobulin from the southern grass frog: absence of binding to the mammalian alpha 2-macroglobulin receptor

An alpha-macroglobulin (alpha 2M), which is a dimer consisting of two non-disulfide-bonded subunits, was identified and purified from frog plasma by Ni2+ chelate affinity chromatography. This frog "half-molecule" alpha-macroglobulin migrated as an alpha 2-globulin in cellulose-acetate electrophoresis rather than as the previously described frog alpha 1M, which exists as a tetramer formed by the noncovalent association of disulfide-bonded pairs. A molecular weight of approximately 380 000 was obtained by gel-filtration high-pressure liquid chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the protein migrated as a single band of Mr approximately 180 000 before and after reduction. No evidence was obtained for association of this protein to a higher molecular weight species. After the preparation was heated, additional bands were obtained in SDS-PAGE with Mr approximately 60 000 and 12 000. The additional bands were not obtained after heating methylamine-treated preparations. The circular dichroic spectrum of frog alpha 2M exhibits negative ellipticity over the region 205-250 nm with a minimum at 216 nm. After reaction with proteinase, a decrease in the absolute mean residue rotation was obtained. Amino acid analysis demonstrated that frog alpha 2M and alpha 1M are similar in composition to avian and mammalian alpha-macroglobulins; however, there are sufficient differences in the composition of these two amphibian alpha-macroglobulins to support the conclusion that they are distinct proteins. Frog alpha 2M bound approximately 0.5 mol of trypsin/mol of inhibitor. This binding was abolished by pretreatment with methylamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for active half-molecules of alpha 2-macroglobulin formed by dissociation in urea

Urea caused dissociation of alpha 2-macroglobulin (alpha 2M) into half-molecules (two disulfide-bonded subunits) as revealed by gel electrophoresis. The fraction of whole molecules remaining decreased with increasing urea concentration. Half-dissociation occurred at about 2.2 M. The ability of alpha 2M to inhibit trypsin also decreased with increasing urea concentration, but the activity-urea curve was shifted to the right as compared to the dissociation-urea curve. Thus, at 3 M urea, gel electrophoresis showed only 6.6% whole molecules, whereas the trypsin inhibitory activity was 95% of that in buffer with no urea, suggesting that half-molecules retain activity. In addition, complexes formed in urea with 125I-labeled trypsin were observed to migrate as half-molecules even though only 50% of such complexes were covalent. These results are surprising in light of the report by Gonias and Pizzo [Gonias, S., & Pizzo, S. (1983) Biochemistry 22, 536-546] that half-molecules formed by mild reduction are active; reduction is assumed to divide the molecule along an axis orthogonal to the break caused by urea. This suggests that active half-molecules can be formed by splitting either the covalent or noncovalent bonds that hold the subunits together. A model is proposed that can account for this possibility. It has the same dimensions and symmetry as a previous model of Feldman et al. [Feldman, S.R., Gonias, S.L., & Pizzo, S.V. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704] and accounts in a similar way for previous functional studies of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of alpha 2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro

When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.     

Functional alpha 2-macroglobulin half-molecules induced by cadmium

Human alpha 2-macroglobulin can be reversibly dissociated by Cd2+ at low ionic strength in half-molecules which retain their ability to bind tightly plasmin and chymotrypsin. The steady state kinetic parameters of these proteinases towards chromogenic substrates when bound to half-molecules are not greatly different from those determined for these enzymes linked to whole alpha 2M molecules. Cd2+ can also induce the dissociation of plasmin- and chymotrypsin - alpha 2M complexes into proteinase-alpha 2M half-molecule conjugates. These results, taken with the fact that monomeric units of alpha 2M cannot bind these proteinases, strongly suggest that each active site of alpha 2M consists in a specific arrangement of two monomeric units linked by disulfide bridges.     

Evolution of human alpha 2-macroglobulin

A papain-binding protein (PBP) resembling human alpha 2-macroglobulin (alpha 2M) but of Mr half that of alpha 2M was purified from plaice (Pleuronectes platessa L.) plasma. The plaice protein displayed most of the distinctive inhibitory properties of the human macroglobulin, and was therefore considered, despite its smaller molecular size, to be homologous with alpha 2M. Plaice PBP was shown to consist of four dissimilar subunits; two I chains (Mr 105 000) and two II chains (Mr 90 000). Each of the larger I chains contained a "bait region" sensitive to proteolytic attack by a variety of proteinases, and an autolytic site analogous to the autolytic site of alpha 2M. Subunit I, almost certainly at the autolytic site, formed SDS-stable, covalent links with methylamine or a proportion of the trapped proteinase molecules. A scheme is proposed for the evolution of human alpha 2M from the smaller fish protein, and the possibility of a shared evolutionary origin for alpha 2M and the complement components C3 and C4 is discussed.     

The electrophoretically ''slow'' and ''fast'' forms of the alpha 2-macroglobulin molecule.

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein...     

Subunit structure of human alpha 2-macroglobulin (alpha 2-MG) with respect to its interaction with trypsin.

SDS-polyacrylamide gel electrophoresis of a recently prepared alpha 2-macroglobulin solution showed only the polypeptide chains of 190,000 molecular weight. Reduction-alkylation of this preparation followed by gel-filtration on a Sephadex G-200 column in 5.2 M guanidine hydrochloride was unable to separate a fraction of 83,000 molecular weight as previously described. Nevertheless, after incubation of a mixture alpha 2-macroglobulin-trypsin during 45 minutes at 37 degrees C, approximately 60 per cent of the preparation were converted in a component with 83,000 molecular weight as detected in SDS polyacrylamide gel. That component was isolated on Sephadex G-200 in guanidine hydrochloride and corresponds to the subunit, fraction II. According to the results of the present work together with those of previous studies, it can be assumed that alpha 2-MG is a 780,000 molecular weight protein (19S) formed of two half-molecules of equal weight (11-12S). The half-molecule contains two polypeptide chains of 180,000-190,000 molecular weight, each of them having, in its middle, a specific region particularly susceptible to attack by proteases.     

Studies on human alpha-2 macroglobulin structure and its complexes with proteases, using polyacrylamide gel electrophoresis]

Pure alpha2M is prepared with fresh plasma as starting material, to prevent the interaction of alpha2M from proteolytic enzymes of plasma such as thrombin, plasmin and kallikrein. During the purification steps, polybrene and aprotin are used as inhibitors and plasminogen is absorbed onto bentonite. When alpha 2M is submitted to polyacrylamide gel electrophoresis (PAA) containing 0.1% SDS, a complete dissociation in two half-molecules of MW 380,000 occurs. When alpha2M is incubated in 1% SDS and 1% beta-mercaptoethanol as reducing agent, only one component of MW 190,000 is observed in PAA-SDS. This experiments show that the alpha2M molecule consist of two symetric halves of same MW (380,000) linked by non covalent bonds. Each two-half-molecules is made of two polypeptides chains MW 190,000 linked by disulfide bonds. Thus alpha2M molecule contains four polypeptides chains having a same MW. The same techniques were applied to the study of alaph2M proteinases complexes. Three different proteinases (plasmin, trypsin and papain) were used in these experiments. Trypsin and papain are commercialy available. Plasminogen was obtained by affinity chromatography and activated into plasmin by insoluble streptokinase fixed on PAB cellulose.     

Characterization of human alpha 2-macroglobulin monomers obtained by reduction with dithiothreitol.

We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1-inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3.     

Reconstitution of urea-dissociated subunits of a pig heart phosphoprotein phosphatase

Pig heart phosphoprotein phosphatase [phosphoprotein phosphophydrolase, EC 3.1.3.16] of Mr 224,000 was dissociated by gel-filtration on Sephacryl S-300, into an active subunit (alpha subunit) of Mr 31,000 and inactive subunits of higher molecular weight in the presence of 6 M urea. After the removal of urea, these subunits reassociated, forming two enzyme forms of Mr 237,000 (Form 1) and Mr 123,000 (Form 2). Form 2 was produced by association of the alpha subunit with an inactive subunit (beta subunit) of Mr 80,000, while Form 1 was formed by combination of the alpha subunit with a complex of inactive subunits which was eluted from a Sephadex G-150 column in fractions of molecular weight range greater than 80,000. The dissociation and reassociation of the subunits of Form 1 by the same urea method produced not only Form 1, but also significant amounts of Form 2, indicating that the inactive subunits of Form 1 were a complex of the beta subunit with another inactive subunit(s). The molecular parameters and other properties of Form 1 were very close to those of the original enzyme. By the conversion of Form 1 to Form 2, the activities of Form 1 towards phosphorylase a and glycogen synthetase b were enhanced 2-3 fold with no significant change in activity towards P-H1 histone or in response to the stimulatory effect of Mg(CH3COO)2 on the dephosphorylation of P-H2B histone. However, removal of the beta subunit from From 2 resulted in strong suppression of activity towards P-H1 histone and response to the salt effect with lesser effects on the activities of Form 2 towards phosphorylase a and glycogen synthase b.     

Physiological levels of binding and iron donation by complementary half-molecules of ovotransferrin to transferrin receptors of chick reticulocytes

Two fragments, each corresponding to approximately half of the ovotransferrin (OTf) molecule and containing an iron-binding site were produced by digestion with affinity bound trypsin and were purified by isoelectric focusing and gel filtration chromatography. The immunologically distinct "half-molecules" individually have little ability to bind to transferrin receptors on chick embryo red blood cells or to donate iron to them. Combining them, however, leads to both binding and iron donation approaching that found for holo-OTf. Furthermore, similar amounts of radiolabeled iron can be extracted into the putative heme fraction from Fe2OTf and from the various combined half-molecules. These findings conflict with those reported by Keung and Azari ( (1982) J. Biol. Chem. 257, 1184-1188) for subtilisin-derived half-molecules of OTf examined in a similar system. They found that each half-molecule appeared to bind at a level of approximately one-third that of Fe2OTf and that the half-molecules competed with each other for binding sites. In contrast, our equilibrium binding studies, in the presence of 2,4-dinitrophenol to prevent iron removal, led to the determination of 4.79 X 10(4) binding sites/cell for Fe2OTf, 4.44 X 10(4) for the NH2-terminal half-molecules in the presence of excess COOH-terminal half-molecules and 4.17 X 10(4) for COOH-terminal half-molecules in the presence of NH2-terminal half-molecules; apparent binding constants were estimated to be 3.29 X 10(6), 1.19 X 10(6), and 0.67 X 10(6) M-1 for these same samples. Problems associated with equilibrium binding studies in which a narrow range of concentrations of ligand is used and/or iron is being removed are discussed. Labeled combined half-molecules were half as effective as labeled Fe2OTf in competition with unlabeled Fe2OTf. These findings are consistent with the lower apparent binding constant found in the equilibrium binding studies. Equimolar apo-OTf had no effect on binding of either Fe2OTf or the combined half-molecules. It seems apparent from our studies that the NH2- and COOH-terminal half-molecules each contain a recognition region both of which are necessary for binding to the transferrin receptor and iron donation to the chick embryo red blood cell.     

Human myeloma IgG half-molecules. Structural and antigenic analyses,.

The structure and antigenic characteristics of a human k, IgG myeloma protein that formed half-molecules were analyzed. Most of the myeloma protein found in the patient's serum and urine consisted to two chain 4.3S half-molecules. A small amount of four chain 7S myeloma protein was, however, found in the serum and was apparently formed by the same clone of tumor cells. Polyacrylamide gel electrophoresis in 8 M urea and 1% sodium dodecyl sulfate and analytical ultracentrifugation in 6 M guanidine of the fully reduced and alkylated half-molecule indicated that this myeloma protein had a heavy chain of a smaller molecular weight (approximately 45,000) than that of normal gamma chains, Except for this apparent deletion, the heavy chain resembled gamma1 chains. The amino acid composition of the peptides containing the half-cysteine residues forming the interchain disulfide bonds, the glycopeptide of the Fc fragment and the COOH-terminal structure were similar if not identical with the analogous structures of gamma1 chains. No Fc fragment could be prepared because the Fc portion of the heavy chain of the myeloma protein was extremely susceptible to degradation with papain. After mild reduction and alkylation, the 7S myeloma protein dissociated into half-molecules, indicating a lack of noncovalent interactions in the Fc fragment that are present in all classes of human immunoglogulins and are responsible for the formation ofFc dimers. The half-molecule was antigenically deficient in the Fc fragment. It failed to precipitate with anti-Fc fragment antisera in double gel diffusion tests and inhibited a Fc-anti-Fc fragment binding reaction weakly and incompletely. The half-molecule and the 7S protein had the same genetic markers on the first and second homology region of the gamma chain. The half-molecule lacked, however, the corresponding markers on the third homology region, These findings suggest that this myeloma protein had a deletion in the gamma chain which was probably located in third homology region and was likely the structural abnormality responsible for the lack of noncovalent interaction in the Fc fragment and absence of most of the antigenic determinants characteristic of gamma chains.     

alpha 2-macroglobulin traps a proteinase in the midregion of its arms. An immunoelectron microscopic study

alpha 2-Macroglobulin, one of the major plasma proteinase inhibitors with Mr = 720,000, is known to inhibit proteinases of all four classes through the "trap mechanism" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724), but the proteinase binding site of alpha 2-macroglobulin has not been identified precisely. We localized bound proteinase molecules on the electron microscopic images of alpha 2-macroglobulin, using anti-proteinase IgG. Serratial Mr = 56,000 proteinase produced by Serratia marcescens was chosen as the antigenic probe in this study because its affinity to specific antibodies was retained in its bound state to alpha 2-macroglobulin. Dimers of alpha 2-macroglobulin/Mr = 56,000 proteinase complexes cross-linked with anti-Mr = 56,000 proteinase IgG were prepared and subjected to electron microscopic observations. The electron microscopic image of alpha 2-macroglobulin complexed with Mr = 56,000 proteinase had four straight arms with an overall shape looking like the character "H." From the way anti-Mr = 56,000 proteinase IgG linked two alpha 2-macroglobulins, it was concluded that the proteinase existed in the midregion of one of the arms. This result helps us to form a more concrete view of the trap mechanism in that one of the arms of alpha 2-macroglobulin wraps the trapped proteinase and holds it isolated from high molecular weight substrates in the surrounding medium.     

Purification and characterization of alpha 1-thiol proteinase inhibitor and its identity with kinin- and fragment 1.2-free high molecular weight kininogen

1. alpha 1-Thiol proteinase inhibitor (alpha 1 TPI) purified from outdated human plasma was a glycoprotein with Mr 83,000 and was composed of heavy and light chains held together with a disulfide bond. 2. The data on amino acid composition, amino terminal sequence of the light chain and carboxyl terminal sequences of the heavy and light chains indicate that alpha 1 TPI is identical with kinin- and fragment 1.2-free HMW kininogen. 3. Purified human plasmin generated a derivative having the same molecular weight (Mr 83,000), same subunit structure (heavy and light chains) and same inhibitory capacity as alpha 1 TPI from HMW kininogen and kinin-free HMW kininogen. This indicated the possibility that alpha 1 TPI is derived from HMW kininogen by plasmin.     

Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.

The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.     

Dissociation of alpha 2 M-macroglobulin into functional half-molecules by mild acid treatment

A slight decrease in pH below neutrality causes the dissociation of alpha 2-macroglobulin (alpha 2M) into dimers formed of two disulfide-bonded subunits. Half-dissociation occurs at pH 6.30 (50 mM NaCl), as determined by gel filtration analysis. The dissociation can be reversed either by increasing the pH or the ionic strength. The ability of alpha 2 M half-molecules at pH 5.75 to bind chymotrypsin is not too different from that of the whole molecule at pH 7.5. Furthermore, the steady-state kinetic parameters toward chromogenic substrate of chymotrypsin bound to alpha 2 M half and whole molecules are quite identical. Likewise, the accessibility of trypsin toward soybean trypsin inhibitor is also fairly similar when involved in half or whole alpha 2 M complexes. These results are consistent with the idea that alpha 2 M-half molecules on chymotrypsin binding undergo a conformational change. This change can be observed by electron microscopy.     

Light-scattering investigation of the subunit structure and dissociation of Helix pomatia hemocyanin. Effects of salts and ureas

The effects of various salts of the Hofmeister and aliphatic acid salt series and hydrophobic reagents of the urea series on the subunit structure and the dissociation of Helix pomatia alpha-hemocyanin were investigated by employing light-scattering molecular weight methods. In moderate ranges of salt concentrations [0-1.0 M NaClO4, NaSCN, NaI, and guanidinium chloride (GdmCl) and 0-2.0 M NaBr], the dissociation reaction is essentially a two-step process characterized by the dissociation of whole hemocyanin molecules dissociating to half-molecules of decamers followed by the dissociation of the half-molecules to five dimeric fragments. The effectiveness of the salts and relative ineffectiveness of the ureas and GdmCl as dissociating agents in the first step of the dissociation reaction suggest that the stabilization of the contact areas between half-molecules in solution is largely a nonhydrophobic energy process involving polar and ionic interactions. Hydrophobic forces appear to be important, however, for stabilization of the half-molecules through side to side contacts of the five dimeric units that make up each half-molecule. The analysis of our dissociation data by use of equations derived in our previous studies [Herskovits, T. T., & Harrington, J.P. (1975) Biochemistry 14, 4964-4971] gave apparent estimates of amino acid groups of about 60-150 for each of the contact areas between the cylindrically shaped half-molecules and 30-60 for each of the dimers in the half-molecules themselves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii.

A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.