Biomedical subjects
Publications and source records attributed to R D Feinman.
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The inhibition of blood platelet aggregation and secretion was studied using covalent thiol reagents, maleimides, or mercuribenzoates, or using inhibitors of protein disulfide isomerase (PDI), bacitracin or antibodies to PDI. As expected, both types of inhibitors were effective against stimulation by normal physiologic stimuli. On the other hand, when stimulation was initiated with the peptide LSARLAF, that specifically activates the integrin alphaIIbbeta3 (the fibrinogen receptor), the PDI inhibitors were without effect. LSARLAF-induced aggregation was, however, inhibited by the sulfhydryl reagents. To further investigate the role of sulfhydryl-containing proteins and alphaIIbbeta3, platelets were labeled with membrane-impermeant sulfhydryl reagents. Nine bands were found labeled on gel electrophoresis. Two of the labeled bands were identified as alphaIIb and beta3. The conclusions are that while PDI is required for platelet aggregation and secretion, an additional sulfhydryl-dependent step or protein is also required. This latter reaction occurs at the level of alphaIIbbeta3. In distinction to most literature reports, at least a subpopulation of alphaIIbbeta3 contains free sulfhydryl groups, consistent with the possibility that it is a substrate for PDI or part of the sulfhydryl-dependent response.
In this study, purified preparations of platelet protein disulfide isomerase (PDI), vitronectin, alpha-thrombin, and antithrombin (AT) were used to demonstrate that PDI catalyzes formation of vitronectin-thrombin-AT complexes. Complex formation requires reduced glutathione (GSH) and can be prevented by N-ethymaleimide, and the formed complex is dissociated by reducing agents such as mercaptoethanol. No vitronectin-thrombin complex formed in the absence of AT, indicating that the thrombin-AT complex is an obligate intermediate in the reaction. Under optimal conditions, the majority of the thrombin-AT is incorporated into the complex in 60 min. Thrombospondin-1, known to form disulfide-linked complexes with thrombin-AT [Milev, Y., and Essex, D. W. (1999) Arch. Biochem. Biophys. 361, 120-126], competes with vitronectin for thrombin-AT in the low-Ca(2+) environment that favors the active form of thrombospondin. The results presented here may also explain previous studies showing that vitronectin-thrombin-AT complexes form better in plasma (which contains PDI) than with purified proteins (where PDI was not used). We were able to purify a PDI from plasma that was immunologically identical to the platelet enzyme. We used the scrambled RNase assay to show that added purified PDI can function in a plasma environment. Complex formation in plasma was inhibited by inhibitors of PDI. PDI was released from the platelet surface in a soluble form at high pH (around the physiologic range), suggesting a source of the plasma PDI. In summary, these studies indicate that PDI functions to form disulfide-linked complexes of vitronectin with thrombin-AT.
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alpha 2-Macroglobulin (alpha 2M) forms several different covalent complexes with proteases. These include unusual forms in which more than one of the four identical subunits of alpha 2M are cross-linked by amide bonds to more than one lysyl amino group of the bound protease. The structure of these complexes and the question of how the identical subunits are arranged to form two protease binding sites are matters of current controversy. The 185-kDa subunits are arranged into two disulfide-bonded half-molecules which are, in turn, noncovalently associated. We have provided evidence that, in the major multivalent cross-linked form, proteases can span the two half-molecules, forming a covalently bonded tetramer [Wang, D., Yuan, A. I., & Feinman, R. D. (1984) Biochemistry 23, 2807-2811]. An alternative theory has recently been proposed in which the major high molecular weight form has two bonds to protease that are within half-molecules--a multivalent cross-linked dimer [Sottrup-Jensen, L., Hansen, H. F., Pedersen, H. S., & Kristensen, L. (1990) J. Biol. Chem. 265, 17727-17737]. To resolve this conflict, experiments were carried out to determine the structure of one of the high molecular weight bands (band 3) seen on SDS-PAGE. Band 3 has anomalous migration, corresponding to markers of apparent molecular mass of 550 kDa (between the tetramer and dimer). In the experiments described here, reactions of thrombin with alpha 2M were run in the presence of methylamine, which competes for one of the two thrombin-alpha 2M covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
An operant chamber has been developed for studying lever-press conditioning in the green crab Carcinus maenas. In one series of experiments, animals were presented with a single bar and were reinforced with food for every bar press. Performance increased over time and high rates of responding were observed after 2 days of training. The response rate was always higher than that for a yoked (noncontingent) control group. When the contingencies were switched, the animals adjusted to the new conditions. Discrimination in the lever-press apparatus was demonstrated in a second experiment in which crabs had to choose between two bars, one (S+) caused food to be dispensed while the other (S-) was inactive. Experimental animals pressed the S+ bar at a significantly higher rate than the S- bar. When the contingencies associated with the lever were reversed, animals learned to switch to the correct bar by the second day. It was not necessary to reinforce every response: animals maintained high rates of responding on a schedule where every other response was reinforced. Animals used different methods of pressing the bar; the most common was extension of the claw, predominantly at the meropodite-carpopodite joint.
Learning in a signaled avoidance procedure was studied in the eye withdrawal reflex of the green crab, Carcinus maenas. A puff of air to the eye, which causes eye retraction, was used as the unconditioned stimulus (US). A mild vibration on the carapace, which has no effect on untrained animals, was used as a warning (conditioned) stimulus (CS). Eye withdrawal during the CS led to the omission of the otherwise scheduled US. Acquisition was rapid, reaching about 75% avoidance after 30 trials. Extinction occurred slowly over the course of 40 CS-only trials. Yoked controls did not perform as well. The behavior of experimental animals in the avoidance procedure was found to be essentially identical to the performance of animals subjected to a classical conditioning paradigm in which CS responses had no effect on US presentation. Additional groups of animals were subjected to experiments in which (a) avoidance conditioning (60 trials) was followed by classical conditioning (40 trials) or (b) classical conditioning was followed by avoidance. The behavior of these groups was, again, essentially identical. The results suggest that there may be an underlying Pavlovian mechanism for the learned response, although the contribution of an operant process is not excluded. The results expand the range of invertebrate animals in which fundamental conditioning phenomena can be demonstrated, and may provide a neuronal model for learning in a signaled avoidance procedure.
Eye withdrawal in the green crab, Carcinus maenas was conditioned by pairing a mild vibration to the carapace as a conditioned stimulus (CS) with a puff of air to one of the eyes as an unconditioned stimulus (US). Animals subjected to repeated pairings showed an increased probability of eye retraction during CS presentation. Significantly less responding was found in several control groups subjected to backward conditioning, unpaired stimuli, stimuli alone, or simply time in the apparatus. Although conditioned animals showed few responses to CS alone after 24 hr, retention could be demonstrated by acquisition that was much more rapid on day 2 than on day 1. Conditioning could also be effected in the eye when it was restrained, a result consistent with reports in the literature that this reflex does not require proprioceptive feedback. Because the neuromuscular circuitry of eye withdrawal is already well defined in Carcinus, this is a promising candidate for studying the neuronal basis of classical conditioning.
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)
Extension of the eye after reflex withdrawal was suppressed by punishing each extension with a brief puff of air. Experimental animals showed a decrease in the rate of responding, and an increase in the latency to the next response during 30-min sessions. The effect of punishment per se was controlled for by the use of yoked animals that received punishments whenever the experimental (master) animals did. This control group did not show the increased latency, and kept the eye erect for most of the session. Experiments were performed with pairs of animals, one eye of each used as master or control, or, alternatively, with single animals in which one eye served as the yoked control for the other. This latter group gave more reliable and reproducible differences between master and yoke than the pairs of animals. Retention was tested by subjecting animals to three sessions separated by a 12-hr rest. The results indicated some savings but this was not a dramatic effect. To demonstrate that the learning was operant in nature, that is, that it depended on the contingency between the act of eye extension and punishment, experiments were performed in which a delay was introduced between the response and the onset of punishments. A delay of 20 s was found to completely eliminate the learned suppression: animals showed latencies close to that of naive animals and responded at a constant high rate throughout the session. Delays of 10, 5, and 2.5 s were found to have a decreasing effect on the learning, and a delay of 1.25 s produced behavior that was within experimental error of that of animals subjected to immediate punishment.
The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.
Complexes formed between thrombin and alpha 2-macroglobulin (alpha 2M) were studied by polyacrylamide gel electrophoresis. The results provide evidence for the existence of a recently proposed novel enzyme-inhibitor species in which a single thrombin molecule forms two or more covalent bonds to two or more different alpha 2M chains. At least one of several slowly migrating bands (greater than 375K on nonreduced gels) that have previously been observed in the literature but not well characterized can be assigned to the new species. The involvement of the lysyl amino groups of thrombin is shown by the observation that methylation of these groups reduces the higher molecular weight bands. In addition, increasing the thrombin:alpha 2M ratio causes a relative decrease in the higher molecular weight species, suggesting that these complexes arise by intramolecular reactions that are susceptible to competition by solution thrombin. The data provide support for our previous proposal [Wang, D., Yuan, A., & Feinman, R.D. (1983) Ann. N.Y. Acad. Sci. 421, 90-97] that the 260K band seen in reduced gels is composed of two proteolyzed inhibitor subunits linked to one thrombin molecule. This intersubunit link maintains the integrity of the alpha 2M in sodium dodecyl sulfate, accounting for the high molecular weight bands under nonreducing conditions. Comparison with a synthetically cross-linked alpha 2M molecule allows a tentative but not unambiguous assignment of one of the bands to this novel structure.
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Experiments were performed to measure the extent to which enzymes bound to alpha 2-macroglobulin (alpha 2M) could be dissociated from the complex. Noncovalent complexes are known to exist between alpha 2M and proteases, such as methyl-trypsin that have had their lysyl amino covalently blocked. Complexes between the inhibitor and native enzymes also have a certain fraction noncovalent binding. Because of the severe steric hindrance imposed on enzymes bound to alpha 2M, even in the noncovalent mode, it has been proposed in the literature that they are not dissociable in the usual sense but, rather, are "trapped" in clathrate-like complexes. The results presented here show that lysyl-blocked methyl-thrombin, or native thrombin are released from their alpha 2M complex by an excess of other lysyl-blocked or native proteases. Under conditions where native thrombin is displaced, labeled enzymes can be incorporated, indicating the inhibitor is intact by the criterion of incorporating enzymes. Likewise, native elastase can be released from its alpha 2M complex by excess cold elastase or the inactive anhydrotrypsin, the latter experiment being carried out with an excess of the low-molecular-weight inhibitor diisopropyl phosphofluoridate. In conjunction with previous results showing that lysyl-blocked enzymes are removed from alpha 2M by soybean trypsin inhibitor, the data indicate that, however sterically hindered, alpha 2M-bound enzymes are dissociable and no unique "trapped" intermediate need be postulated.
The analysis of thrombin-alpha 2M reaction mixtures by two-dimensional SDS-PAGE has allowed us to assign several probable molecular species to the mixture of complexes formed. These include structures previously described in, or predicted from, the literature, as well as two types of novel species: Divalent cross-linking of two inhibitor chains by a single enzyme molecule. Very high molecular weight species that are attributed to intermolecular cross-linking of more than one inhibitor molecule. Species containing enzyme monovalently linked to an intact subunit are not supported by our data, but are not excluded and additional study will be required to determine if they exist.
The primary observation, from our laboratory and others, of the effect of blocking the lysyl amino groups of enzymes is the reduction in the fraction of complexes that are resistant to SDS. The blocked enzyme derivatives do cause the specific proteolysis of the alpha 2M subunit to the 85K/100K fragments, and do cause the appearance of new thiol groups. With respect to the sequence of reaction, we may summarize the results by saying that if the reversible DMM-trypsin is, in fact, a model for the native enzyme, proteolysis can precede formation of the presumed covalent bond between bound enzyme and inhibitor. If our preliminary observations are borne out by later experiments, thiol release may precede covalent bond formation or loss of reactivity with amines, suggesting that an intact thiolester need not be the immediate target for amines; another intermediate, possibly the internal pyroglutamate originally proposed by Howard et al. and seen in model studies, may be an additional, or even the primary, target for covalent bonding with native enzymes. With regard to the "trap" hypothesis, the limited release of thiols in a slow phase is suggestive of enzyme activity within the alpha 2M-protease complex, consistent with the theory. Noncovalent irreversible complexes, however, are not a necessary part of associations seen with lysyl-blocked enzymes (which do cause proteolysis and do release thiols); this result is supported by limited data with noncovalently bound native enzymes. Some fraction of irreversible noncovalently bound enzymes may occur, but our results suggest that although alpha 2M-bound enzymes are unusually sterically hindered, the transformation to the presumed covalent state that appears to depend on intact amino groups, may be sufficient to explain the low dissociation of native enzymes. We feel that more experimental evidence is needed to resolve some of the ambiguities on this question but, we feel the existence of a "trapping" reaction has not been proved. In fact, given the possible existence of equilibria between covalent and noncovalent complexes observed, for example, in soybean trypsin inhibitor, and the very low dissociation constants observed with traditional protein-protein complexes, the question of physically encapsulated structures in alpha 2M may not be resolvable without direct evidence from crystal structures.
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