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L V Favreau

Publications and source records attributed to L V Favreau.

23 records · Page 2Linked to original sources
PubMed1982

Purification and properties of human platelet heparitinase.

An endoglycosidase which cleaves heparin and heparan sulfate was isolated from outdated human platelets by freeze-thaw solubilization, heparin-Sepharose chromatography, DEAE-cellulose chromatography, hydroxylapatite chromatography, octyl-agarose chromatography, concanavalin A-Sepharose chromatography, and Sephacryl S-200 gel filtration. The overall extent of purification of the platelet heparitinase is about 240,000-fold and the overall yield of the enzyme is about 5.6% as compared to the initial freeze-thaw solubilization preparation. The final product is physically homogeneous as judged by disc gel electrophoresis at acidic pH as well as gel filtration chromatography and exhibits an apparent molecular weight of approximately 134,000. Furthermore, our results indicate that the above enzyme is present within platelet lysosomes. The biologic potency of the endoglycosidase was examined as a function of pH. The data show that the platelet heparitinase is maximally active from pH 5.5 to pH 7.5. However, the enzyme possesses minimal ability to cleave heparin at pH less than 4.0 or greater than 9.0. The substrate specificity of the platelet endoglycosidase was determined by identifying susceptible linkages within the heparin molecule that can be cleaved by the above component. Our studies indicate that this enzyme is only able to hydrolyze glucuronsylglucosamine linkages. Furthermore, investigation of the structure of the disaccharide which lies on the nonreducing end of the cleaved glucuronic acid residue suggests that N-sulfation of the glucosamine moiety or ester sulfation of the adjacent iduronic acid groups are not essential for bond scission.

Blood Platelets↗
PubMed1982

Inhibition of vascular smooth muscle cell growth by endothelial cell-derived heparin. Possible role of a platelet endoglycosidase.

Bovine aortic endothelial cells release a heparin-like substance in the presence of 0.4% fetal calf serum. This substance inhibited the growth of smooth muscle cells in vitro by about 70%. Substitution of platelet-poor plasma for serum resulted in minimal liberation of inhibitory activity from the cells unless at least 10-fold higher concentrations of platelet-poor plasma were utilized. This suggested that a platelet product was involved in the release process. Therefore, we examined the ability of the platelet heparitinase described in the preceding communication to release heparin-like species from cultured endothelial cells. Our results show that when endothelial cells were exposed to serum-free medium containing 1 ng/ml of the purified platelet endoglycosidase, at least as much inhibitory activity was released as was obtained with 0.4% serum. Dose response experiments indicated that only 10 pg/ml of the enzyme were necessary to liberate 50% of the inhibitory activity from endothelial cells. The heparin-like nature of the inhibitory substance was demonstrated by its sensitivity to Flavobacterium heparinase. Utilizing appropriate controls, the release of heparin-like material by the endoglycosidase was shown to be enzyme-specific and was not due to artifacts of experimental manipulations. In addition, this enzyme did not convert prereleased material to an active component, but directly liberated the active heparin-like species from endothelial cells. A simple model describing the possible role of heparin-like components and the endoglycosidase in the normal and injured wall is presented.

Animals↗
PubMed1982

Heparin with two binding sites for antithrombin or platelet factor 4.

A new, highly discriminating affinity chromatographic technique has been developed which employs antithrombin and concanavalin A-Sepharose to fractionate heparin species of all molecular sizes. This methodology is able to subdivide the active mucopolysaccharide pools of molecular weight 6,000 to 8,000 (LMW) or 18,000 to 22,000 (HMW) into various species with descending affinities for antithrombin as well as decreasing anticoagulant potencies. The upper 10% of these two pools, either LMW or HMW highly active heparin, appears to be relatively homogeneous with respect to interactions with antithrombin and possessed anticoagulant potencies of 350 units/mg and 731 units/mg, respectively. The HMW highly active heparin has been examined by analytic ultracentrifugation. It exhibited a charge-connected weight-average molecular weight of 22,000 +/- 2,000 with minimal size heterogeneity. The stoichiometries of interaction of antithrombin and platelet factor 4 with HMW highly active heparin as determined by fluorescence spectroscopy indicated that 2 molecules of either protein are able to bind to 1 molecule of the mucopolysaccharide. These studies also reveal that the binding of antithrombin to HMW highly active heparin is characterized by KDISSHAT = 5.0 X 10(-8) M and KDISSHAT2 = 1.0 x 10(-7) M, respectively. The avidity of platelet factor 4 for HMW highly active heparin could not be quantitated but appears to be at least 10 to 100 times greater than that of antithrombin for mucopolysaccharide.

Antithrombins↗
PubMedDate not supplied

The interaction of cytochrome b5 with four cytochrome P-450 enzymes from the untreated rat.

The capacity of four native P-450 enzymes to interact with cytochrome b5 was compared and discussed in terms of spin shift and metabolism. Two-dimensional electrophoresis was employed as a tool to aid in characterization of the different enzymes isolated from liver microsomes of the untreated rat. RLM5 had a pl of 7.4 and RLM3 had a pl of 7.1. Two new forms isolated by this laboratory, RLM2 and RLM5a, differed from other forms of cytochrome P-450 characterized to date. The pl values of these forms were 7.35 and 7.6, respectively. The interaction of all four enzymes with cytochrome b5 differed. Cytochrome b5 caused a major low to high spin transition when added to RLM5. The latter hemoprotein was 28% high spin at 25 degrees C and was shifted to 55% high spin by cytochrome b5. RLM5a shifted from 4% high spin to 15% high spin under comparable conditions. In contrast, RLM2 and RLM3 were both minimally influenced by cytochrome b5, reaching only 8% high spin. Cytochrome b5 did not appreciably influence the rates of metabolism of aminopyrine, benzphetamine, testosterone, or p-nitroanisole with RLM2 or RLM3. However, with RLM5 and RLM5a, rates of aminopyrine and benzphetamine demethylation and testosterone hydroxylation were increased to about 130% with RLM5 and up to 200% with RLM5a. The demethylation of p-nitroanisole was stimulated by cytochrome b5, 3.5-fold with RLM5 and 14-fold with RLM5a. In no case was the ratio of monohydroxy metabolites of testosterone altered by the addition of cytochrome b5, indicating an effect on Vmax rather than Km.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗