Inactivation of human plasma alpha 1-antichymotrypsin by microbial proteinases

Incubation of human plasma alpha 1-antichymotrypsin with proteinases from various microbial sources resulted in the enzymatic inactivation of the inhibitor as determined by loss of inhibitory activity against alpha-chymotrypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reaction products indicated that intact alpha 1-antichymotrypsin (Mr 67000) had been converted to an inactive form (63000) by limited proteolysis. No stable proteinase/inhibitor complexes were detected, and no random proteolysis of the inactivated inhibitor occurred even after prolonged incubation with the proteinases. Metallo- and serine proteinases from several microbial sources all readily inactivated alpha 1-antichymotrypsin. Since alpha 1-antichymotrypsin is also an early stage acute phase reactant, its inactivation may be important in disrupting bodily defense mechanisms.     

The receptor-binding domain of human alpha 2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase

Limited proteolysis of human alpha 2-macroglobulin (alpha 2M) by a novel bacterial proteinase resulted in the isolation of a soluble 20-kDa domain. The isolated fragment contained the receptor recognition site, expressed on alpha 2M complexes, as it competed effectively with alpha 2M-trypsin for binding to the receptor on skin fibroblasts. The fragment also reacted with two monoclonal antibodies which define epitopes that are part of the receptor recognition site. Characterization of the 20-kDa domain showed it to contain an intact disulfide bridge, while its susceptibility to N-glycanase and reaction with concanavalin A indicated the presence of N-linked carbohydrate. The NH2-terminal sequence (Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Glu-Thr-Leu-Pro-Glu-Thr-Cys-Asp-Glu -Pro) proved this fragment to constitute the COOH terminus of human alpha 2M. Proteolysis occurred at Lys1313-Glu which together with the observation that tosyllysine chloromethyl ketone was an effective inhibitor of the bacterial proteinase, would indicate the latter to hydrolyze preferentially peptide bonds carboxyl-terminal to lysine residues.     

Analysis of the effects of snake venom proteinases on the activity of human plasma C1 esterase inhibitor, alpha 1-antichymotrypsin and alpha 2-antiplasmin

Incubation of C1 esterase inhibitor with Crotalid, Viperid and Colubrid snake venoms resulted in enzymatic inactivation of the inhibitor. Intact inhibitor (104 kDa) was converted into an active intermediate species of 89 kDa and then a further cleavage resulted in formation of an 86-kDa inactive inhibitor. In contrast, C1 esterase inhibitor did not lose activity during incubation with Elapid venoms; however, the intact inhibitor was gradually converted to an active species of 89 kDa during the incubation. Human alpha 1-antichymotrypsin was inactivated by all venoms tested, including those from the Elapid family. The 67-kDa intact inhibitor was converted by the venom proteinases to an inactive 63-kDa form. The results suggest that this acute-phase plasma protein is readily susceptible to inactivation by venom proteinases. Human alpha 2-antiplasmin (68 kDa) was cleaved to form a 61-kDa active intermediate, which then underwent a second cleavage to produce an inactive 53-kDa product. Elapid venoms had no effect on alpha 2-antiplasmin activity and did not cleave this inhibitor. All inhibitors were inactivated with catalytic amounts of venom proteinases. No stable proteinase-proteinase inhibitor complexes were detected, and no random proteolysis of the inhibitors occurred.     

Purification and some properties of two proteinases from Crotalus adamanteus venom that inactivate human alpha 1-proteinase inhibitor.

Two proteinases (proteinases I and II) have been purified from Crotalus adamanteus venom to the stage of electrophoretic homogeneity and proteinase II has been crystallized. The proteinase differ slightly in molecular weight and amino acid composition. Both are metalloenzymes requiring Zn2+ or Ca2+, or both; neither requires thiol compounds for activation. The proteinases are free of esterolytic activity against benzoly-L-arginine ethyl ester and benzoyl--tyrosine ethyl ester. Proteinase II cleaves the oxidized B chain of insulin at the bonds Phe1-Val2, His5-Leu6, His10-Leu11, Ala14-Leu15, Leu15-Tyr16, and Tyr-16-Leu17. Digestion of polylsine and polyarginine by proteinase II liberates products ranging from dodecapeptides to hexapeptides. Proteinases I and II catalytically inactive human plasma alpha 1-proteinase inhibitor (54,000 daltons). Electrophoretic analysis of the reaction of proteinase II with alpha 1-proteinase inhibitor reveals that an inactivated inhibitor species of 50,000 daltons is formed, and a peptide of 4,000 daltons is released. The gradual disappearance of the native inhibitor results in the corresponding loss of inhibitory activity against trypsin and chymotrypsin.     

Alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, or alpha(2)-macroglobulin is required for vascular smooth muscle cell spreading in three-dimensional fibrin gel

It is assumed that vitronectin and other adhesion molecules induce cell spreading. We found that vascular smooth muscle cells require unidentified plasma components besides adhesion molecules to spread in fibrin gel, a likely provisional matrix at wound sites. By purification, the plasma components were found to be alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, and alpha(2)-macroglobulin. The chemically inactivated alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin lose the spreading activity, indicating that these proteins function as proteinase inhibitors but not as adhesion molecules. Not only anti-integrin (alpha(v)beta(3) and alpha(5)beta(1)) antibodies but also anti-fibronectin antibodies inhibit the cell spreading. The spreading occurs without the addition of fibronectin and integrins, suggesting that cells produce these molecules. In the absence of the proteinase inhibitors, Western blot analysis shows that the fibronectin is degraded in fibrin gel, while it is intact in the presence of the inhibitors. Thus, the proteinase inhibitors prevent adhesion molecules such as fibronectin from being degraded by a cell-derived proteinase(s) and thus play a role in cell spreading.     

The reaction of serpins with proteinases involves important enthalpy changes

When active serpins are proteolytically inactivated in a substrate-like reaction, they undergo an important structural transition with a resultant increase in their conformational stability. We have used microcalorimetry to show that this conformational alteration is accompanied by an important enthalpy change. For instance, the cleavage of alpha(1)-proteinase inhibitor by Pseudomonas aeruginosa elastase, Staphylococcus aureus V8 proteinase, or papain and that of antithrombin by leukocyte elastase are characterized by large enthalpy changes (DeltaH = -53 to -63 kcal mol(-1)). The former reaction also has a large and negative heat capacity (DeltaC(p)() = -566 cal K(-1) mol(-1)). In contrast, serpins release significantly less heat when they act as proteinase inhibitors. For example, the inhibition of pancreatic elastase, leukocyte elastase, and pancreatic chymotrypsin by alpha(1)-proteinase inhibitor and that of pancreatic trypsin and coagulation factor Xa by antithrombin are accompanied by a DeltaH of -20 to -31 kcal mol(-1). We observe no heat release upon proteolytic cleavage of inactive serpins or following inhibition of serine proteinases by canonical inhibitors or upon acylation of chymotrypsin by N-trans-cinnamoylimidazole. We suggest that part of the large enthalpy change that occurs during the structural transition of serpins is used to stabilize the proteinase in its inactive state.     

Conformational modulation of purified glycoprotein (GP) IIb-IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb-IIIa.

This study characterized conformational states of platelet glycoprotein IIb-IIIa (GPIIb-IIIa) and regions of the molecule required for fibrinogen binding. Platelet lysates were passed sequentially over concanavalin A and aminoethylglycine (Aeg)RGDS affinity columns. Approximately 10% of the total GPIIb-IIIa bound to the Aeg-RGDS column. The non-binding GPIIb-IIIa was further purified by S300 gel filtration. Only GPIIb-IIIa which recognized immobilized RGDS bound fibrinogen. The functional difference between the Aeg-RGDS binding GPIIb-IIIa (active) and the S300-purified complex (inactive) suggested that the two populations existed in different conformations. This was confirmed immunochemically and in an assay utilizing endoproteinase Arg-C. Active GPIIb-IIIa was heavily degraded by Arg-C, whereas inactive GPIIb-IIIa was highly resistant to degradation. Receptor occupancy by RGDV or peptidomimetic inhibitors prevented degradation of regions of the active complex and stimulated hydrolysis of the inactive receptor such that the two populations yielded fragments of identical electrophoretic mobility. Induction of hydrolysis of inactive GPIIb-IIIa required 15-fold higher concentrations of RGDV than protection of the active complex. Upon removal of inhibitor, fragments generated from either active or inactive GPIIb-IIIa bound fibrinogen. The ability of carboxypeptidase Y to digest inhibitor-protected GPIIb-IIIa was also examined. GPIIb was cleaved to a 58-kDa NH2-terminal fragment, whereas GPIIIa remained essentially intact. The complexed fragments bound fibrinogen with similar affinity as intact GPIIb-IIIa. This binding was inhibited by both RGDV and HHLGGAKQAGDV peptides. These data suggest that: 1) purified active and inactive GPIIb-IIIa exist in different conformations and have different affinities for RGDV; 2) certain peptidomimetic inhibitors (Ro 42-1499 and Ro 43-5054) alter the conformation of inactive GPIIb-IIIa; 3) GPIIIa and a 58-kDa NH2-terminal fragment of GPIIb alpha form a high affinity fibrinogen binding complex.     

Inactivation of blood plasma alpha 1-proteinase inhibitor as affected by 2 splenic thiol proteinases active in a neutral medium

It has been found that two active in neutral medium thiol proteinases from bovine spleen, cathepsin L and cathepsin H, bring about rapid and irreversible inactivation of alpha 1-proteinase inhibitor (alpha 1PI)--one of the major plasma inhibitors of serine proteinases. The activity of the enzymes studied did not change upon the interaction with alpha 1PI. With stoichiometric proteinase/inhibitor ratio, the inactivation of alpha 1PI under the effect of cathepsin L was instantaneous, while under the effect of cathepsin H it occurred within 30-60 min. The products of alpha 1PI inactivation had an inhibitory effect on the rate of its reaction with cathepsin L. alpha 1PI inactivation under the action of cathepsin L and cathepsin H was accompanied by the decrease in the molecular mass of the inhibitor from 54 kDA to 46 kDa. This was, probably, caused by the hydrolysis of the peptide bond formed by NH2 group of threonine. The 46 kDa fragment did not undergo further degradation. It did not bind to immobilized trypsin but retained antigenic properties. The results obtained show that the limited proteolysis is a mechanism of the inhibitor inactivation. It is suggested that under some conditions thiol proteinases, upon their release from the cell, participate in the control of effective alpha 1PI concentration.     

Amino acid sequence of derivatives of newborn rat epidermal thiol proteinase inhibitor

Three different forms of thiol proteinase inhibitor (TPI) were isolated from newborn rat epidermis, in which two forms, TPI-1 and TPI-2, inhibited a proteinase activity, but another newly detected one (designated as TPI-3), showed no inhibitory effect. The complete amino acid sequence of TPI-2 and the sequence of the first seventeen residues from the NH2-terminus of TPI-3 were determined. The sequence shows that TPI-2 lacks in the first six (or four) residues from the NH2-terminus of intact inhibitor, TPI-1, whereas TPI-3 is devoid of its fifteen amino acid residues. These results indicate a high and specific susceptibility of TPI to proteolysis. Most significantly, the NH2-terminal region of TPI appears to be essential for inhibition of proteinase activity.     

Selective activation of MAPK(erk1/2) by laminin-1 peptide alpha1:Ser(2091)-Arg(2108) regulates macrophage degradative phenotype

Components of the extracellular matrix contain cryptic domains, which are exposed by proteolysis and elicit biological responses distinct from intact molecules. The disparate cellular response to extracellular matrix fragments and parent intact molecules suggests differential recognition and signaling pathways. In experiments reported here, we demonstrate that urokinase and matrix metalloproteinase-9 expression by RAW264.7 macrophages is stimulated by a synthetic laminin peptide derived from the alpha1-chain (SRARKQAASIKVAVSADR), whereas intact laminin-1 has no effect on proteinase expression by macrophages. Incubation of macrophages with alpha1:SRARKQAASIKVAVSADR stimulates tyrosine phosphorylation of several proteins including mitogen-activated protein kinase (MAPK)(erk1/2). In contrast, neither intact laminin-1 nor the beta1-chain peptide CDPGYIGSR stimulated protein tyrosine phosphorylation in these cells. Inhibition of tyrosine kinases or protein kinase C blocked alpha1-chain peptide-induced phosphorylation of MAPK(erk1/2) and the up-regulation of steady state levels of urokinase mRNA and matrix metalloproteinase-9 activity. A MAPK kinase inhibitor blocked alpha1-chain-induced phosphorylation of MAPK(erk1/2) and the induction of proteinase expression. Intact laminin-1, which was unable to induce macrophage proteinase expression, failed to stimulate the phosphorylation of MAPK(erk1/2). These data demonstrate that incubation of macrophages with alpha1:SRARKQAASIKVAVSADR, but not intact laminin-1, triggers protein kinase C-dependent activation of MAPK(erk1/2), leading to the up-regulation of proteinase expression.     

Independent analysis of bait region cleavage dependent and thiolester bond cleavage dependent conformational changes by cross-linking of alpha 2-macroglobulin with cis-dichlorodiammineplatinum(II) and dithiobis(succinimidyl propionate)

Treatment of the human plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) with proteinase results in conformational changes in the inhibitor and subsequent activation and cleavage of the internal thiolester bonds of alpha 2M. Previous studies from this laboratory have shown that cross-linking the alpha 2M subunits with cis-dichlorodiammineplatinum(II) (cis-DDP) prevents the proteinase-induced conformational changes which lead to the activation and cleavage of the internal thiolester bonds of alpha 2M. In addition, cis-DDP treatment prevents the proteinase- or CH3NH2-induced conformational changes in alpha 2M which lead to a "slow" to "fast" change in nondenaturing polyacrylamide gel electrophoresis. In this paper, we demonstrate that treatment of alpha 2M with dithiobis(succinimidyl propionate) (DSP) also results in cross-linking of the subunits of alpha 2M with concomitant loss of proteinase inhibitory activity. Although proteinase is not inhibited by DSP-treated alpha 2M, bait region specific proteolysis of the alpha 2M subunits still occurs. Unlike cis-DDP-treated alpha 2M, however, incubation of DSP-treated alpha 2M with proteinase does not prevent the bait region cleavage dependent conformational changes which lead to activation and cleavage of the internal thiolester bonds in alpha 2M. On the other hand, cross-linking of alpha 2M with DSP does prevent the conformational changes which trigger receptor recognition site exposure following cleavage of the alpha 2M thiolester bonds by CH3NH2. These conformational changes, however, occur following incubation of the CH3NH2-treated protein with proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.     

The inactivation of human plasma alpha 1-proteinase inhibitor by proteinases from Staphylococcus aureus

The interaction of three proteinases (seryl, cysteinyl, and metallo-) from Staphylococcus aureus with human plasma alpha 1-proteinase inhibitor has been investigated. As expected, none of the enzymes was inactivated by this protein, each, instead causing the conversion of the native inhibitor into an inactive form of decreased molecular weight. Amino-terminal sequence analysis indicated that inhibitor inactivation had occurred by peptide bond cleavage near the reactive center of this protein. When the inhibitor was modified by this treatment, it became resistant to both pH and temperature denaturation and, in contrast to the intact denatured protein, did not undergo further proteolytic degradation. This process of inactivation of alpha 1-proteinase inhibitor by pathogenic proteinases could result in a deregulation of its target enzyme, neutrophil elastase, and, therefore, may be important in the consumption of some plasma proteins by this enzyme during septicemia.     

alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.     

The reactive site of human alpha 2-antiplasmin

Human alpha 2-antiplasmin rapidly forms a stable, equimolar complex with either its target enzyme, plasmin, or with trypsin. Perturbation of the inhibitor-trypsin complex results in peptide bond cleavage at the reactive site of the inhibitor with the concomitant release of a small peptide fragment which apparently represents the carboxyl-terminal segment of the inhibitor. Sequence analysis of this fragment, together with that of an overlapping peptide obtained by treatment of native inhibitor with either Staphylococcus aureus V8 proteinase or human neutrophil elastase, yields data which indicate that the reactive site of alpha 2-antiplasmin encompasses a P1-P'1 Arg-Met sequence. However, unlike alpha 1-1-proteinase inhibitor which has a Met residue in the P1-position, oxidation of alpha 2-antiplasmin has no effect on its inhibitory activity toward either plasmin, trypsin, or chymotrypsin, indicating the lesser mechanistic importance of the P'1-residue during enzyme inactivation by this inhibitor.     

Assay of alpha-cysteine proteinase inhibitor in serum or plasma

A new proteinase inhibitor has recently been found in human serum or plasma which specifically inhibits cysteine proteinases such as ficin, papain, bromelain and cathepsin B. However, serum contains alpha 2-macroglobulin which also inhibits these cysteine proteinases and, consequently, interferes with the assay of the new alpha-cysteine proteinase inhibitor. Therefore, assay of the inhibitor in serum has not been established previously. In the present method, the alpha 2-macroglobulin is inactivated by preincubating the serum in methylamine solution at 55 degrees C, while the alpha-cysteine proteinase inhibitor retains its activity. The inhibitory power against cysteine proteinases is found to be due mainly to this protein in human serum. This inhibitor is also found in mammals such as cows, pigs and rats. Vitamin E deficient rats show a very high inhibitor level. Therefore, the present method will enable us to investigate the relation between diseases and the activity of the alpha-cysteine proteinase inhibitor. Also, this method is simple and inexpensive. The necessary amount of serum is only 10 microliter.     

Kinetics of association of human proteinases with human alpha 2-macroglobulin

Association rates have been determined for the interaction of human alpha 2-macroglobulin with human neutrophil elastase, cathepsin G, and human plasma kallikrein. Both of the neutrophil enzymes are rapidly inactivated by this inhibitor; however, the inactivation of plasma kallikrein is much slower. Comparison of the rates of inactivation with those already established for other inhibitors clearly indicate that alpha 1-proteinase inhibitor is the controlling inhibitor for neutrophil elastase and alpha 1-antichymotrypsin for cathepsin G, alpha 2-macroglobulin acting only as a secondary inhibitor. The control of plasma kallikrein would appear to be rather poor since neither alpha 2-macroglobulin nor C1-inhibitor appears to react very rapidly with this proteinase. Thus, a primary role for alpha 2-macroglobulin in directly inactivating proteinases in blood, under normal physiological conditions, remains to be established.     

Limited proteolysis of the alpha-macroglobulin rat alpha 1-inhibitor-3. Implications for a domain structure.

Rat alpha 1-inhibitor-3 is a 180-kDa monomeric proteinase inhibitor found in high concentration in rat plasma. By several criteria it has been shown to be a member of the family of alpha-macroglobulin proteinase inhibitors often exemplified by the tetrameric human alpha 2-macroglobulin. We have used limited proteolysis of rat alpha 1-inhibitor-3 to probe the domain structure of this family of proteins. Proteinases of different specificities, including trypsin, chymotrypsin, thermolysin, and Staphylococcus aureus V8 proteinase, were employed and a common fragmentation pattern was observed when the reaction products were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These fragments were electrotransferred to polyvinylidene difluoride membranes and subjected to NH2-terminal amino acid sequence analysis in order to position them within the context of the primary structure. The fragmentation pattern may define the domain structure of alpha 1-inhibitor-3 and serve as a model for the domain organization of the family of alpha-macroglobulin proteinase inhibitors.     

Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.     

The structure of alpha 2-macroglobulin-methylamine after papain digestion as determined by electron microscopy.

alpha 2-Macroglobulin-methylamine (alpha 2M-CH3NH2) was digested with papain at pH 5.0. The major 600 kDa fragment was purified by molecular-exclusion chromatography. In a non-denaturing gel-electrophoresis system, the 600 kDa fragment migrated in a single band at a rate that was comparable with that for the untreated alpha 2M-CH3NH2. The elution volume of the 600 kDa fragment on Superose-6 was slightly increased. In primary cultures of rat hepatocytes, cellular uptake of 125I-alpha 2M-CH3NH2 was not affected by the 600 kDa fragment, confirming the results of other investigators. The 600 kDa fragment was negatively stained with uranyl formate and analysed by transmission electron microscopy. The major structural characteristics of the parent protein (alpha 2M-CH3NH2) remained intact. The most common image included prominent lateral walls and two centrally located regions of stain exclusion termed 'paddle structures'. The distance between the paddle structures was equivalent in alpha 2M-CH3NH2 and the 600 kDa fragment [approximately 13.5 nm (135 A)]. By contrast, the lateral walls in the 600 kDa fragment were decreased in length by approximately 0.37 nm (37 A) (19%). It is proposed that the 600 kDa structure retains the 'hollow cylinder' shape of alpha 2M-CH3NH2. The structure of the cylinder is formed by the lateral walls and four paddle structures (only two are imaged, owing to overlapping). The paddle structures in the 600 kDa fragment are intact and relatively closer to the apices of the molecule, owing to the decrease in lateral wall length. Since the alpha 2M receptor-binding sites are removed by papain digestion, the studies presented here support the location of the receptor-binding sites near the apices of the lateral walls.