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C W Pratt

Publications and source records attributed to C W Pratt.

29 records · Page 2Linked to original sources
PubMed1988

Antithrombin action of phosvitin and other phosphate-containing polyanions is mediated by heparin cofactor II.

We have examined the antithrombin effects of various phosphate-containing polyanions (including linear polyphosphates, polynucleotides and the phosphoserine glycoprotein, phosvitin) on the glycosaminoglycan-binding plasma proteinase inhibitors, antithrombin III (ATIII) and heparin cofactor II (HCII). These phosphate-containing polyanions accelerate the HCII-thrombin reaction, as much as 1600-fold in the case of phosvitin. The HCII-thrombin reaction with both phosvitin and polynucleotides appears to follow the ternary complex mechanism. The HCII-thrombin complex is rapidly formed in the presence of these phosphate polyanions (each at 10 micrograms/ml) when 125I-labeled thrombin is incubated with human plasma (ex vivo). None of these phosphate polyanions accelerate the ATIII-thrombin reaction. Our results suggest that the antithrombotic effect of these phosphate-containing polyanions is mediated by HCII activation and not by ATIII.

Antithrombin III↗
PubMed1988

In vivo catabolism of heparin cofactor II and its complex with thrombin: evidence for a common receptor-mediated clearance pathway for three serine proteinase inhibitors.

The plasma clearance of 125I-labeled human heparin cofactor II and its complex with thrombin was studied in mice to determine whether a specific mechanism exists for the catabolism of the inhibitor-proteinase complex. Initial studies demonstrated that murine plasma contains a heparin cofactor II-like inhibitor as shown by the presence of a dermatan sulfate-sensitive thrombin inhibitor. Human heparin cofactor II cleared from the circulation of mice with an apparent half-life of 80 min while heparin cofactor II-thrombin complexes cleared with an apparent half-life of only 10 min. The specificity of the clearance mechanism was investigated by clearance competition studies involving coinjection of excess unlabeled heparin cofactor II-alpha-thrombin, antithrombin III-alpha-thrombin, or alpha 1-proteinase inhibitor-elastase, and by tissue distribution studies. The results demonstrated that the clearance of 125I-labeled heparin cofactor II-alpha-thrombin is a receptor-mediated process, and that the same hepatocyte receptor system recognizes complexes containing heparin cofactor II, antithrombin III, and alpha 1-proteinase inhibitor.

Animals↗
PubMed1987

The role of inter-alpha-trypsin inhibitor and other proteinase inhibitors in the plasma clearance of neutrophil elastase and plasmin.

The plasma clearance of neutrophil elastase, plasmin, and their complexes with human inter-alpha-trypsin inhibitor (I alpha I) was examined in mice, and the distribution of the proteinases among the plasma proteinase inhibitors was quantified in mixtures of purified inhibitors, in human or murine plasma, and in murine plasma following injection of purified proteins. The results demonstrate that I alpha I acts as a shuttle by transferring proteinases to other plasma proteinase inhibitors for clearance, and that I alpha I modulates the distribution of proteinase among inhibitors. The clearance of I alpha I-elastase involved transfer of proteinase to alpha 2-macroglobulin and alpha 1-proteinase inhibitor. The partition of elastase between these inhibitors was altered by I alpha I to favor formation of alpha 2-macroglobulin-elastase complexes. The clearance of I alpha I-plasmin involved transfer of plasmin to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Results of distribution studies suggest that plasmin binds to endothelium in vivo and reacts with I alpha I before transfer to alpha 2-macroglobulin and alpha 2-plasmin inhibitor. Evidence for this sequence of events includes observations that plasmin in complex with I alpha I cleared faster than free plasmin, that plasma obtained after injection of plasmin contained a complex identified as I alpha I-plasmin, and that a murine I alpha I-plasmin complex remained intact following injection into mice. Plasmin initially in complex with I alpha I more readily associated with alpha 2-plasmin inhibitor than did free plasmin.

Alpha-Globulins↗
PubMed1987

Mechanism of action of inter-alpha-trypsin inhibitor.

Inter-alpha-trypsin inhibitor (I alpha I) is a unique proteinase inhibitor that can be proteolyzed by the same enzymes that are inhibited, to generate smaller inhibitors. This study examines the reactions of I alpha I with trypsin, chymotrypsin, plasmin, and leukocyte elastase. Complexes of I alpha I and proteinase were demonstrated by gel filtration chromatography. Complete digestion of I alpha I by each proteinase was not accompanied by a comparable loss of inhibition of that enzyme or a different enzyme. Following proteolysis, inhibitory activity was identified in I alpha I fragments of molecular weight 50,000-100,000 and less than 40,000. Addition of a second proteinase inhibitor prevented proteolysis. Both I alpha I and its complex with proteinase were susceptible to degradation. Kinetic parameters for both the inhibition and proteolysis reactions of I alpha I with four proteinases were measured under physiological conditions. On the basis of these results, a model for the mechanism of action of I alpha I is proposed: Proteinase can react with either of two independent sites on I alpha I to form an inhibitory complex or a complex that leads to proteolysis. Both reactions occur simultaneously, but the inhibitory capacity of I alpha I is not significantly affected by proteolysis since the product of proteolysis is also an inhibitor. For a given proteinase, the inhibition equilibrium constant and the Michaelis constant for proteolysis describe the relative stability of the inhibition and proteolysis complexes; the second-order rate constants for inhibition and proteolysis indicate the likelihood of either reaction. The incidence of inhibition or proteolysis reactions involving I alpha I in vivo cannot be assessed without knowledge of the exact concentrations of inhibitor and proteinases; however, analysis of inhibition rate constants suggests that I alpha I might be involved in plasmin inhibition.

Alpha-Globulins↗
PubMed1987

Structure-function relationships in heparin cofactor II: spectral analysis of aromatic residues and absence of a role for sulfhydryl groups in thrombin inhibition.

This study characterizes the structural and functional significance of sulfhydryl residues in human plasma heparin cofactor II (HCII). For quantification of sulfhydryl groups, the extinction coefficient of HCII was redetermined and found to be 0.593 ml mg-1 cm-1 using second-derivative spectroscopy and multicomponent analysis assuming 4, 10, and 2 residues of tryptophan, tyrosine, and tyrosine-O-sulfate per mole of protein, respectively. The results show that tyrosine-O-sulfate residues in HCII and in cholecystokinin peptide fragments (as model compounds) do not significantly contribute to the absorbance spectrum from 280 to 300 nm. A total of three sulfhydryl groups per mole of HCII was detected by Ellman's reagent titration, with or without treatment with dithioerythritol, indicating the absence of intramolecular disulfide bonds. Incubation of HCII with 0.1-10 mM dithioerythritol did not diminish its heparin-enhanced thrombin inhibition activity. Treatment with various sulfhydryl-specific reagents, including p-mercuribenzoate, HgCl2, and N-substituted maleimide derivatives, inactivated HCII. Titration with Ellman's reagent after these reactions identified the modification site as a cysteinyl residue(s). However, complete methanethio derivatization of the sulfhydryl groups of HCII using methyl methanethiosulfonate did not alter heparin-catalyzed thrombin inhibition. These results indicate that the sulfhydryl groups of HCII are not essential for thrombin inhibition. HCII differs from antithrombin III, which contains an essential disulfide bond for heparin-dependent thrombin inhibition (Longas, M. O., et al. (1980) J. Biol. Chem. 255, 3436). Furthermore, within the "serpin" (serine proteinase inhibitor) superfamily, HCII resembles chicken ovalbumin in occurrence of sulfhydryl residues and reactivity with various sulfhydryl group-directed compounds.

Chloromercurinitrophenols↗
PubMed1986

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.

Alpha-Globulins↗
PubMed1984

The effect of zinc and other divalent cations on the structure and function of human alpha 2-macroglobulin.

Zinc binding to human alpha 2-macroglobulin was studied to assess its involvement in the structure and function alpha 2-macroglobulin. Equilibrium dialysis experiments indicated multiple classes of zinc-binding sites, the one of highest affinity having a site number of 20 and a Kd value of 8 X 10(-7) M. Native alpha 2-macroglobulin and alpha 2-macroglobulin-trypsin complexes bound comparable amount of zinc. The proteinase inhibitory activity of alpha 2-macroglobulin was not affected by zinc binding at physiological concentrations nor by the removal of zinc by EDTA. Above 25 microM zinc, alpha 2-macroglobulin activity decreased, although binding of [125I]trypsin was not affected. When nondenaturing gel electrophoresis was performed, the preparation of alpha 2-macroglobulin migrated as half-molecules at increasing zinc concentration. Experiments with other divalent cations correlated decreases in alpha 2-macroglobulin activity with apparent dissociation of the alpha 2-macroglobulin tetramer in the presence of copper and mercury, but not barium, cadmium or nickel. While zinc binding to alpha 2-macroglobulin does not function in proteinase inhibition, it might be involved in zinc transport in vivo. At nonphysiological concentrations, zinc and other divalent cations are useful as probes of protein quaternary structure.

Binding Sites↗
PubMed1984

Identification of "embryonin" as bovine alpha 2-macroglobulin.

Pedersen fetuin contains a contaminant, previously named "embryonin" that exhibits immuno-cross reactivity with human alpha2-macroglobulin (alpha2Mh). In the present study, it is demonstrated that this protein coelutes with alpha2Mh in gel filtration chromatography and can be purified to homogeneity by Zn2+ chelate chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this contaminant exhibited similar subunit size, protease-induced cleavage fragments, and heat fragmentation as alpha2Mh. 125I-trypsin and 125I-chymotrypsin each bound at a ratio of 0.9 mol/mol to this fetuin-derived native alpha2M (alpha2Mf) and at a ratio of less than 0.2 mol/mol to methylamine-treated alpha2Mf. As determined by SDS-PAGE, 1:1 molar ratio of protease to alpha2Mf cleaved each alpha2Mf subunit to fragments of Mr approximately 72,000. At a 0.2:1 molar ratio of trypsin to alpha2Mf-methylamine, every alpha2Mf-methylamine subunit was cleaved to polypeptide chains of Mr approximately 72,000 and 110,000. In native PAGE, alpha2Mf and alpha2Mf-methylamine migrated with the same mobility; after reaction with trypsin their mobilities increased similarly. 125I-alpha2Mf cleared from the circulation of mice with a t1/2 of 30 min. The trypsin or methylamine derivative of 125I-alpha2Mf cleared with t1/2 of less than 5 min and clearance was competable when the ligand was co-injected with a large molar excess of unlabeled alpha2Mh-methylamine. alpha2Mf, 0.3 nM, treated with trypsin or methylamine, inhibited 50% of the binding of 0.1 nM 125I-alpha2Mh-methylamine to specific receptors on mouse peritoneal macrophages in vitro. Native alpha2Mf did not inhibit significantly the binding of the ligand at this concentration. Bovine alpha2M (alpha2Mb) was purified from plasma by Ni2+ chelate chromatography. By SDS-PAGE, amino acid analysis, and cyanogen bromide peptide mapping, it was indistinguishable from the alpha2M purified from fetuin. It is concluded that embryonin is bovine alpha2M.

Amino Acids↗
PubMed1984

Conformation-specific precipitation of human alpha 2-macroglobulin by divalent zinc or calf thymus histone H3.

Highly purified native alpha 2-macroglobulin (alpha 2M), alpha 2M-trypsin, and alpha 2M-methylamine were compared in experiments designed to study protein precipitation. Significant turbidity developed within 30 min in solutions containing histone H3 and either alpha 2M-methylamine or alpha 2M-trypsin, as determined by absorbance at lambda = 550 nm. No turbidity was detected in solutions that contained histone H3 and native alpha 2M or histone H3 alone. Experiments with radioiodinated histone H3 or radioiodinated proteinase inhibitor confirmed that both the H3 and the alpha 2M "fast" forms (alpha 2M-methylamine, alpha 2M-trypsin) were present in the precipitates generated. As much as 70% of the 125I-alpha 2M-methylamine was recovered in the precipitate after incubation with a 120-fold molar excess of H3 (concentration of alpha 2M-methylamine, 0.28 microM). The ratio of histone to proteinase inhibitor by weight in the precipitate was approximately two. Under comparable conditions, somewhat less alpha 2M-trypsin precipitated from solutions containing H3 than did alpha 2M-methylamine; however, inactivation of the alpha 2M-trypsin with phenylmethylsulfonyl fluoride prior to incubation increased the level of precipitation significantly. Solutions containing poly-L-lysine (Mr approximately 13,000) instead of histone did not form precipitates with any of the forms of alpha 2M studied. In a second set of experiments, radioiodinated native alpha 2M, alpha 2M-trypsin, and alpha 2M-methylamine were incubated in solutions containing ZnCl2, BaCl2, CdCl2, CuSO4, MgCl2, or NiCl2 (concentration of divalent cation between 5 microM and 1.0 mM). Native alpha 2M was soluble in all of these salts. By contrast, alpha 2M-methylamine and alpha 2M-trypsin precipitated extensively from solutions containing greater than 100 microM ZnCl2. Precipitation was greater than 90% complete at 1 mM ZnCl2. A similar effect was not observed with any of the other divalent cations.

Cations, Divalent↗
PubMed1977

Escherichia coli ribosomal protein S1 has two polynucleotide binding sites.

The interaction of Escherichia coli ribosomal protein S1 with a variety of RNA and DNA oligomers and polymers has been studied, using both a sedimentation technique and the quenching of intrinsic protein fluorescence upon nucleic acid binding to obtain equilibrium binding parameters. Two polynucleotide binding sites have been detected on S1: site I binds either single-stranded DNA or RNA and does not discriminate between adenine- and cytidine-containing polynucleotides, while the II binding is highly specific for RNA over DNA and shows a marked preference for cytidine polynucleotides over the corresponding adenine-containing species. On the basis of the binding properties of S1 to denatured DNA cellulose and poly(rC)-cellulose, it is demonstrated that every S1 molecule carries both a site I and a site II. Some possible implications of these results for mechanisms of protein synthesis and phage Qbeta replication are briefly considered.

Bacterial Proteins↗