Antioxidant activity of probucol and its analogues in hypercholesterolemic Watanabe rabbits.

Probucol (1) and probucol analogues with the substitutions at the disulfide-linked carbon (2, 3) and an additional substitution at a tert-butyl of each phenolic ring (4) were tested for their ability to lower total serum cholesterol and prevent aortic atherosclerosis in modified Watanabe heritable hyperlipidemic (WHHL) rabbits and to inhibit Cu2(+)-induced lipid peroxidation of isolated plasma low-density lipoproteins (LDL). After 84 days of feeding 1% of each compound in rabbit chow, probucol was effective in lowering serum cholesterol, whereas 2-4 were not. The concentration of drug in serum and LDL was 2 greater than 1 greater than 3 greater than 4. Probucol and analogues prevented Cu2(+)-induced oxidation of LDL in vitro to an extent that directly related to their concentrations in LDL. The decrease in lipid oxidation was directly correlated with the inhibition of both oxidized-LDL-induced cholesteryl ester synthesis in cultured macrophages and to the inhibition of aortic atherosclerosis in vivo. These results show that the antioxidant activity of probucol and analogues is directly related to their concentration in LDL, which may explain their pharmacological activity in reducing atherosclerosis.

Characterization of a monoclonal antibody specific to the amino terminus of the alpha-chain of human fibrin.

A peptide, Gly-Pro-Arg-Val-Val-Glu, corresponding to the first six residues of the amino terminus of the alpha-chain of human fibrin (desAA-fibrin) was prepared by solid-phase peptide synthesis. The peptide was covalently linked to keyhole-limpet hemocyanin (KLH) and used as an immunogen for preparing monoclonal antibodies. A monoclonal antibody specific to the hexapeptide, but not to KLH or fibrinogen, was produced. The antibody did not bind to thrombin-mediated clots prepared from either plasma or purified fibrinogen. However, immunoreactivity was detected when fibrin (prepared from fibrinogen) was solubilized with 8 M urea. In contrast, a monoclonal antibody specific to the amino terminus (Gly-His-Arg-Pro-Leu-Asp-Lys) of the beta-chain of fibrin recognized the epitope in clots. These results indicate that thrombin cleavage of fibrinogen produces a structural change in the amino terminal domain of the alpha-chain that makes it inaccessible to antibody interaction. In addition, our study suggests that the potential clinical application of monoclonal antibodies to localize fibrin-rich thrombi must take into account the final structure of clots.

Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase.

Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.

Monoclonal antibodies that distinguish between active and inactive forms of human postheparin plasma hepatic triglyceride lipase.

Hepatic triglyceride lipase (H-TGL) was purified from human postheparin plasma. Specific monoclonal antibodies (MAbs) were produced that discriminate between active (native) and inactive (denatured) forms of the enzyme. Mice immunized with native H-TGL resulted in MAbs that recognized only the native protein. The antibodies did not react with H-TGL treated with 1% sodium dodecyl sulfate or heated at 60 degrees C. The loss of immunoreactivity with heating correlated directly with the loss of enzyme activity and there was a corresponding increase in immunoreactivity with the MAbs prepared against the denatured enzyme. Western blot analysis of postheparin plasma with the MAbs against denatured H-TGL gave a single protein band of 65 kD; preheparin plasma showed no detectable immunoreactivity with either MAb. These immunochemical studies suggest that there are no circulating active or inactive forms of H-TGL in man. Furthermore, the MAbs provide the necessary reagents for development of immunoassays for H-TGL.