alpha-chymotrypsin and Hyperlipidemias

The apoprotein (apoB) of low density lipoprotein (LDL) is reported to be a large polypeptide, and it is proposed that there are two similar-sized subunit proteins in LDL (Smith, Dawson, and Tanford. 1972. J. Biol. Chem. 247: 3376-3381.). When apoB is isolated under conditions that minimize artifactual proteolysis, only a single, large molecular weight protein appears on polyacrylamide gel electrophoresis in SDS. To investigate the organization of apoB as it exists within native LDL, limited proteolysis with trypsin has been used as a structural probe. Tryptic digestion for 1 hr at pH 7.6 with enzyme-to-protein ratios of 1:100 and 1:5 results in the liberation of approximately 10% and 30% of apoB as smaller, water-soluble peptides. These peptides may be separated from the partially digested but still intact tryptic core (T-core) of the lipoprotein by chromatography on Sephadex G-75. Repeatedly, the 1:5 T-core of native LDL is found to contain a family of polypeptides of 14,000-100,000 molecular weight. Although they have lost significant quantities of apoprotein, these T-cores sustain an appearance of homogeneity, as studied by analytical ultracentrifugation. Their measured molecular weights do not differ appreciably from those of the native LDL, and the carbohydrate content of the 1:5 tryptic T-core of LDL is similar to that of the native LDL. In normolipemic individuals, LDL generally exists in a monodisperse state, but, in different individuals, monodisperse LDL may range in molecular weight from 2.4 to 3.9 x 10(6). Limited tryptic digestions were used to probe the organization of apoB in these different molecular weight LDL. As assayed by SDS-acrylamide gel electrophoresis of the larger polypeptides and fingerprinting of the smaller released peptides, those regions of LDL exposed to trypsin digestion are identical in monodisperse LDL of 2.5 and 3.4 x 10(6) molecular weight. Thus, the different quantities of lipid bound in these various LDL must interact with apoB so that the same regions of the apoprotein are exposed to the action of trypsin in these different molecular weight lipoproteins.

Topics: Amino Acids; Apolipoproteins; Carbohydrates; Chymotrypsin; Humans; Hyperlipidemias; Lipoproteins, LDL; Molecular Weight; Pronase; Trypsin

C-I was prepared from very low density lipoproteins of patients with familial type V hyperliporproteinemia. Peptides from tryptic digests of unmodified and succinylated C-I, chymotryptic peptides, and the products of cayanogen bromide cleavage were isolated and characterized. Sequence analysis of tryptic peptides was performed by the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) technique and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. Alignment of the tryptic fragments within the cyanogen bromide and succinyl-tryptic peptides was confirmed by the overlap chymotryptic peptides. The complete amino acid sequence of C-I, 57 residues in length, does not reveal any obvious basis for its lipophilic properties.

Topics: Amino Acid Sequence; Amino Acids; Aminopeptidases; Apoproteins; Carboxypeptidases; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Thin Layer; Chymotrypsin; Cyanogen Bromide; Dansyl Compounds; Humans; Hyperlipidemias; Lipoproteins, VLDL; Peptide Fragments; Peptide Hydrolases; Serine; Trypsin

Topics: Amino Acid Sequence; Amino Acids; Aminopeptidases; Animals; Apoproteins; Carboxypeptidases; Chromatography, Gas; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Thin Layer; Chymotrypsin; Cyanogen Bromide; Electrophoresis, Polyacrylamide Gel; Humans; Hyperlipidemias; Immunodiffusion; Lipoproteins, VLDL; Peptide Fragments; Rabbits; Serine; Spectrophotometry; Spectrophotometry, Ultraviolet; Trypsin; Ultracentrifugation

Topics: Acute Disease; Adult; Amylases; Cholecystokinin; Cholesterol; Chylomicrons; Chymotrypsin; Exocrine Glands; Female; Humans; Hyperlipidemias; Lipoproteins; Male; Middle Aged; Pancreas; Pancreatic Diseases; Pancreatitis; Secretin; Triglycerides

Topics: Alcoholism; Amylases; Anemia, Hemolytic; Cholecystokinin; Chymotrypsin; Clinical Enzyme Tests; Fatty Liver; Humans; Hyperlipidemias; Jaundice; Pancreas; Secretin; Trypsin