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Biomedical subjects

R D Feinman

Publications and source records attributed to R D Feinman.

At least 37 records · Page 2Linked to original sources
PubMed1982

beta-Adrenergic response in mouse CNS reaggregate cultures.

Primary reaggregate cultures from 17-day embryonic mouse brains were prepared and tested for beta -adrenergic response. Isoproterenol stimulated the production of cAMP 3- to 4-fold over basal levels after 10 days in culture. The response to isoproterenol was rapid, reaching a maximum 1-2 min after stimulation, and was blocked by propranolol. The beta-adrenergic receptor was characterized in membranes prepared from reaggregates by measurement of specific binding of [125I]iodohydroxybenzylpindolol (IHYP). The Kd for IHYP was 100-300 pM and did not change as a function of time in culture. A maximum receptor density of approximately 80-110 fmol/mg membrane protein was found. Both beta-adrenergic receptors and response appeared at times in culture, which were approximately equivalent to birth.

Adrenergic beta-Agonists↗
PubMed1981

Inhibition of proteases by alpha 2-macroglobulin. The role of lysyl amino groups of trypsin in covalent complex formation.

The lysyl amino groups of bovine trypsin were covalently modified by acetylation, succinylation, or reductive methylation. The enzymatically active derivatives were still capable of reaction with alpha 2-macroglobulin (alpha 2M), although to a lesser extent than native enzyme. The resulting enzyme-alpha 2M complexes, however, were much more susceptible to dissociation by sodium dodecyl sulfate than complexes formed with unmodified trypsin. The bound modified enzymes could be released from the alpha 2M complex with an excess of native thrombin. In addition, anhydrotrypsin displaced methyl trypsin from its complex and the anhydro derivative was bound in its place. The data provide evidence for two types of noncovalent intermediates; those formed from lysyl-modified enzymes show proteolysis of the alpha 2M to the nominal 85,000 fragment, whereas anhydrotrypsin forms a complex with apparently intact alpha 2M chains. A model is proposed for the reaction of alpha 2M with proteases in which one or both of these noncovalent intermediates is formed. Conversion of this form(s) to a stable covalent complex requires unmodified lysyl amino groups on the enzyme, suggesting that these groups may form a covalent bond with the inhibitor, possibly at the site at which methylamine binds.

Binding Sites↗
PubMed1980

Reaction of antithrombin with proteases. Evidence for a specific reaction with papain.

Experiments were performed to determine if the sulfhydryl protease, papain (EC 3.4.22.2), reacts with the plasma protease inhibitor antithrombin (antithrombin III, heparin cofactor) on a specific manner analogous to the reaction of thrombin (EC 3.4.21.5) and other serine proteases with this inhibitor. The esterolytic activity of papain is blocked by the addition of antithrombin, but not by antithrombin-thrombin complex or by protein substrates such as bovine serum albumin. Likewise, in the presence of papain, antithrombin was unable to displace the active site dye proflavine from thrombin, or to inhibit thrombin-catalysed hydrolysis of an anilide substrate. The reaction of antithrombin and papain was not accelerated by low concentrations of heparin. Approximately stoichiometric amounts of heparin completely inhibited the reaction of papain with antithrombin. The mutual inhibition indicates that plasma antithrombin does react with papain but the reaction differs from the interaction with coagulation factors, particularly in the heparin effect.

Antithrombin III↗
PubMed1979

Antithrombin reactions with alpha- and gamma-thrombins.

Human alpha-thrombin with high clotting activity and its proteolyzed derivative gamma-thrombin with virtually no clotting activity reacted in an essentially identical manner with antithrombin. The two enzyme forms bound proflavin with similar constants and showed identical behavior with small substrates. No significant differences were found for the antithrombin reactions (measured by proflavin displacement or active site titration) with respect to kinetics, extent of reaction, or effect of added heparin. The enzyme--antithrombin complexes could not be dissociated with sodium dodecyl sulfate (NaDodSO4) but the NaDodSO4-denatured complexes were dissociated by hydroxylamine treatment. The gamma-thrombin-antithrombin complex has an approximate molecular weight of 75 000 by disc gel electrophoresis as compared with 100 000 for the alpha-complex, consistent with the polypeptide structures of the two proteins. The gamma-thrombin--antithrombin complex did not inhibit clotting catalyzed by alpha-thrombin. In addition, fibrinogen did not affect the reaction of gamma-thrombin with antithrombin or antithrombin--heparin. Thus, the antithrombin and antithrombin--heparin reactions do not involve the fibrinogen recognition sites which are destroyed by proteolytic conversion of alpha-thrombin to the noncoagulant gamma form.

Antithrombins↗
PubMed1978

Evidence that calcium regulates platelet function.

It is generally believed that calcium ions play a key role in regulation of platelet function. This is based on 3 types of evidence. 1. Analogies with other cells. Calcium ions are known to trigger secretion and contraction in many cells, possibly reflecting a general role for calcium in all secretion and contraction. 2. Indirect evidence. Platelet aggregation and secretion are induced by divalent cation ionophores. The response to the ionophore A23187 is identical to that induced by other potent stimuli. 3. Direct evidence. Platelet activation can be blocked by drugs (e. g. certain local anesthetics) that block release of calcium ions from sarcoplasmic reticulum; the inhibition can be overcome by addition of extracellular calcium in the presence of a calcium ionophore. While this does not constitute definitive proof, the central role for calcium ions remains an attractive hypothesis that justifies attempts to further define calcium pools and fluxes in platelets.

Blood Platelets↗
PubMed1977

Quantitative evaluation of the loss of human platelet dense bodies following stimulation by thrombin or A23187.

1. Distributions of dense bodies per platelet were evaluated in unstimulated human platelets and in platelets stimulated to secrete by the addition of varying amounts of thrombin or the ionophore A23187. Since distributions were not Gaussian, they were compared by non-parametric methods. 2. Most distribution obtained following varying levels of dense-body release differed significantly from the control (unstimulated) distribution. In general, distribution from points widely separated in terms of per cent release of total dense-body content also differed significantly. Distributions obtained at points where the total number of dense bodies lost was approximately the same did not differ significantly, regardless of the drug and incubation conditions used to produce the dense-body loss. 3. Dense bodies were lose from platelets during the secretory process (measured as the release of either calcium or ATP), and the number of dense bodies lost correlated at a significant level with the amounts of both substances secreted. 4. Following maximal secretion produced by either thrombin or A23187, a population of dense bodies (refractory dense bodies) remained in platelets. The refractory dense bodies could not have been distributed uniformly among the control populations of either platelets or dense bodies. The distribution of refractory dense bodies could, however, be described by a model in which platelets containing large numbers of dense bodies had a greater proportion of their dense-body complement refractory than did platelets containing fewer dense bodies.

Adenosine Triphosphate↗
PubMed1977

Interrelations of platelet aggregation and secretion.

The mechanism of stimulus-response coupling in human platelets was investigated with a new instrument that simultaneously monitors aggregation and secretion in the same sample of plateletrich plasma. When platelets were stimulated by high concentrations of ADP, secretion began only after aggregation was almost complete. With lower concentrations of ADP or with epinephrine, biphasic aggregation was observed, and secretion began simultaneously with, or slightly after, the second phase of aggregation. When platelets were stimulated with high concentrations of gamma-thrombin or A23187, secretion and aggregation began essentially together. With very low concentrations of gamma-thrombin or A23187, biphasic aggregation was observed with secretion paralleling the second phase. At every concentration of collagen, secretion and aggregation appeared to be parallel events. Under every condition where the beginning of secretion lagged behind aggregation, secretion was dependent upon aggregation and was inhibited by indomethacin; this is referred to as aggregation-mediated platelet activation. When secretion began at the same time as aggregation, it also occurred in the absence of aggregation and was not blocked by indomethacin; this is referred to as directly induced platelet activation. These observations are consistent with a simple model of platelet stimulus-response coupling that includes two mechanisms for activation; aggregation-mediated activation is inhibited by indomethacin, while direct activation does not depend upon aggregation and is not inhibited by indomethacin. Secretion and second wave aggregation appear to be parallel events, with little evidence for second wave aggregation being a consequence of secretion as usually described.

Adenosine Diphosphate↗
PubMed1977

The lumi-aggregometer: a new instrument for simultaneous measurement of secretion and aggregation by platelets.

We have developed a new instrument that simultaneously monitors aggregation and secretion of ATP from the same sample of platelets. Aggregation is determined by the usual turbidimetric method; secretion of ATP is followed by measuring luminescence from the firefly luciferase system at right angles to the aggregometer light path. Data are recorded on a two-pen strip chart recorder and reveal the temporal relations between the two platelet responses.

Adenosine Triphosphate↗
PubMed1976

Specificity of thrombin: evidence for selectivity in acylation rather than binding for p-nitrophenyl alpha-amino-p-toluate.

The lysyl ester analogue p-nitrophenyl alpha-amino-p-toluate hydrobromide was synthesized, and its reactions with thrombin, trypsin, and plasmin were studied by stopped-flow and conventional methods. Kinetic parameters were compared with those determined for the arginyl ester analogue, p-nitrophenyl p-guanidinobenzoate hydrochloride, with these enzymes. By following nitrophenol release or proflavin absorption changes in the stopped-flow spectrophotometer, the constants Ks (enzyme-substrate binding), k2 (acylation), and k3 (deacylation) were determined. The major findings were: (1) Ks values were similar regardless of the substrate or the enzyme; (2) k3 was approximately the same for the reaction of the lysyl ester analogue with any enzyme; (3) k2 for the lysyl ester analogue was 1100 times greater with trypsin than with thrombin; and (4) k2 with thrombin was 60 times greater for the arginyl than for the lysyl ester analogue. The results suggest that the limited cleavage of lysyl bonds by thrombin is due in part to restricted acylation rather than substrate binding. The active site of thrombin, compared with that of trypsin, appears to have a more stringent requirement for the spatial relationship between the cationic group and the bond cleaved in substrates.

Acylation↗