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J C Holt

Publications and source records attributed to J C Holt.

49 records · Page 3Linked to original sources
PubMed1983

Effect of heparin on the in vivo release and clearance of human platelet factor 4.

Intravenous injection of heparin (100 U/kg) into normal volunteers resulted in an increase of platelet factor 4 (PF4) level in platelet-poor plasma from a mean value of 18.1 +/- 6.6 ng/ml before the injection to 257.9 +/- 68.3 ng/ml at 5 min after injection. PF4 antigen isolated from "postheparin plasma" by adsorption on heparin-agarose and elution with 2.0 M NaCl and "authentic PF4" isolated from human platelets showed identical patterns of migration as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Material released by washed human platelets was injected intravenously into rats. The clearance of PF4 followed a biphasic exponential pattern. The half-lives (T1/2) for the fast and slow components for control rats were 1.2 and 17.1 min. Heparin significantly extended the half-life of human PF4 in rat circulation. The clearance of PF4 injected together with heparin followed a single component model with a half-life of 27.6 min. Administration of heparin to rats that had been previously injected with human platelet releasate resulted in a 30-fold increase of plasma PF4 level in their circulation. The clearance of PF4 from the circulation of these rats (T1/2 = 45 min) fitted a single component model. We propose that PF4 is originally secreted by platelets into circulation and subsequently bound reversibly to vascular sites from which it can be released back into the circulation by heparin. The fast component of PF4 clearance that is abolished by heparin may reflect binding of this protein to the endothelial cells.

Animals↗
PubMed1982

Human platelet basic protein. Its relation to low affinity platelet factor 4 and beta-thromboglobulin.

Human beta-thromboglobulin, low affinity platelet factor 4 and platelet basic protein have been purified to homogeneity from the material released by thrombin-stimulated platelets. Purification steps included isoelectric focusing and heparin-agarose chromatography. Antibodies against each of these proteins have been raised in rabbits. Antigenic identity of the proteins has been demonstrated in radioimmunoassay using 125I-labelled platelet basic protein or 125I-labelled low affinity platelet factor 4 and a variety of antibodies. The molecular weight of platelet basic protein estimated by gel filtration in 6 M guanidine hydrochloride using Sepharose 6B corresponded to approx. 10 000 daltons, slightly higher than that of beta-thromboglobulin (8851 daltons) and low affinity platelet factor 4 (9278 daltons). These findings raise the possibility that the formation of low affinity platelet factor 4 beta-thromboglobulin may be a consequence of the action of proteolytic enzymes on platelet basic protein.

Amino Acid Sequence↗
PubMed1981

Factor VIII-related activities in therapeutic concentrates.

Recent studies have suggested that factor VIII exists in plasma as a complex containing two distinct antigens (VIII R:Ag and VIII C:Ag) and two different biological activities (VIII:C and VIII R:RCo). Concentrations of these four entities have been measured in all the different types of therapeutic material used for treatment of hemophilia and von Willebrand's disease. In all materials, the ratio of VIII R:Ag to VIII:C was greater than 1: however, there were significant differences between concentrates, and the lowest ratio was found in cryoprecipitate. In VIII R:Ag assays, the freeze-dried concentrates gave nonparallel dose-response curves compared with plasma, indicating alterations in molecular form during purification. Values for VIII C:Ag were much lower than the corresponding VIII R:Ag values, and ratios of VIII C:Ag to VIII:C were close to 1 in many of the concentrates, suggesting that the low yields of VIII:C in some production processes could result from losses of VIII:C molecules rather than from denaturation. Measurement of VIII R:RCo by a platelet counting method gave lower values in most concentrates than by an aggregometry technique. It is proposed that the platelet counting method may reflect closely the biological activity of the concentrates in treatment of von Willebrand's disease.

Antigens↗
PubMed1980

Conversion of low-affinity platelet factor 4 to beta-thromboglobulin by plasmin and trypsin.

Low-affinity platelet factor 4 and beta-thromboglobulin are platelet-secreted proteins that bind with low affinity to heparin. They show extensive immunological cross-reactivity and appear to differ in amino acid sequence only by an amino-terminal peptide unique to low-affinity platelet factor 4. The possibility that beta-thromboglobulin is derived from low-affinity platelet factor 4 by proteolysis was investigated by exposing this protein to the action of plasmin, thrombin and trypsin. While thrombin had no effect, plasmin and trypsin converted low-affinity platelet factor 4 to a species with the same electrophoretic mobility and isoelectric point as beta-thromboglobulin. We conclude that beta-thromboglobulin is a breakdown product of low-affinity platelet factor 4.

Amino Acid Sequence↗
PubMed1977

Distribution of alkali light chains in myosin: isolation of isoenzymes.

Antibodies have been isolated which are specific for the "difference peptide" unique to the alkali 1 light chain (mol wt 20 700) of chicken breast muscle myosin. When coupled to Sepharose as an immunoadsorbent, they are capable of resolving subfragment 1, heavy meromyosin, and myosin into two fractions, one rich in alkali 1 and the other rich in alkali 2. This fractionation provides direct evidence for the existence of two isoenzymic populations in vertebrate skeletal myosin. The ability of antibodies to the difference peptide to distinguish between alkali 1 and 2 provides a marker which will allow the distribution of alkali light chains in muscle fibers and filaments to be investigated.

Animals↗
PubMed1975

An immunological approach to the role of the low molecular weight subunits in myosin. I. Physical--chemical and immunological characterization of the light chains.

The light chains of chicken breast muscle myosin (alkali 1 and 2, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) 1.c.) have been isolated in pure form and characterized with respect to amino acid composition, uv and circular dichroism (CD) spectral properties, and molecular weight. Antibodies specific for each of the light chains have been used to demonstrate the similarity of alkali 1 and 2 (mol wt 21,000 and 16,000, respectively), and the distinctness of these from DTNB 1.c. (mol wt of 18,000). The DTNB 1.c. isolated by a variety of methods were all immunologically identical. Significant cross-reactivity was observed between corresponding rabbit and chicken light chains, confirming other indications of homology between these proteins in the two species. The immunological difference between alkali 1 and 2 was largely accounted for by an N-terminal peptide, rich in proline, alanine, and lysine, which is unique to alkali 1. The presence of antibodies to this peptide in anti-alkali 1 serum suggests an immunological approach to the question of how alkali 1.c. are distributed in myosin.

Amino Acids↗
PubMed1975

An immunological approach to the role of the low molecular weight subunits in myosin. II. Interaction of myosin and its subfragments with antibodies to the light chains.

Immunological methods, in parellel with measurement of ATPase activity, have been used to characterize the reactions of antibodies specific for light chains with myosin and its water-soluble proteolytic subfragments, heavy meromyosin (HMM) and subfragment 1 (HMM S-1). Antiserum to the 5,5'-dithiobis(2-nitro-enzoic acid) (DTNB) light chain undergoes a precipitation reaction with all of the enzyme species, in which half of the homologous light chain is selectively dissociated. The results suggest that the incomplete dissociation reflects the way in which the light chain is bound, rather than the existence of two distinct species of DTNB 1.c. Little reaction was observed with antisera to alkali-released light chains, indicating that these components in myosin and the subfragments are either largely buried or else conformationally different from the isolated light chains used as immunogens. None of the antisera produced significant changes in Ca2+- or EDTA-ATPase activities. Moreover, calcium regulation through the troponin-tropomyosin system was unaffected by removal of DTNB 1.c. from myosin, as well as from the subfragments. The absolute level of actin-activated ATPase activity was, however, consistently lower in the presence of light chain antisera (or purified IgG and antibody) than in aqueous buffer or nonimmune serum. For both alkali and DTNB 1.c. antisera, this loss in activity seemed to result from steric hindrance of actin binding by antibody bound to undissociated light chain. Experimental conditions which would be expected to weaken such an antigen-antibody interaction, as well as the use of monovalent Fab in place of IgG, decreased the inhibition of activity. Altogether the activity measurements suggest that the light chains, particularly DTNB 1.c., are probably not integral parts of either the hydrolytic or actin-binding sites.

Actins↗
PubMed1972

Studies of the denaturation and partial renaturation of ovalbumin.

1. The denaturation of ovalbumin by the reagents sodium dodecyl sulphate and guanidinium chloride was investigated, by following the changes in sedimentation velocity, optical rotatory dispersion and viscosity as a function of denaturant concentration. 2. With sodium dodecyl sulphate both the optical-rotatory-dispersion parameters a(0) and b(0) become more negative, the sedimentation coefficient decreases and the viscosity increases; significant differences in the denaturation profiles are observed. The change in each parameter is indicative of only limited denaturation. 3. With guanidinium chloride the transition occurs over the concentration range 1-4m: more extensive changes occur in all the physical parameters than with sodium dodecyl sulphate. The values of a(0) and b(0) are indicative of complete denaturation. Reduction by mercaptoethanol produces only minor further changes. 4. Renaturation was attempted from both denaturants, the removal of reagent being accomplished reversibly by controlled slow dialysis. Partial renaturation was observed, but aggregated or insoluble material was produced in both cases at relatively low concentrations of denaturant. Similar behaviour was observed with fully reduced protein in guanidinium chloride-mercaptoethanol; complete renaturation could not be brought about even at very low protein concentrations.

Dialysis↗