carbobenzoxycarbonyl-l-phenylalanyl-l-alanine-d-diazomethane and leupeptin

Topics: Animals; Caseins; Culture Media; Cysteine Proteinase Inhibitors; Diazomethane; Endopeptidases; Leucine; Leupeptins; Lipoproteins; Trichomonas vaginalis

The effects of in situ postrigor injection (24 h postmortem) of exogenous aspartic, serine, and cysteine proteinase effectors into cylindrical beef longissimus samples on tenderness and myofibrillar protein degradation and integrity were studied. Injection of phenylmethanesulphonylfluoride (PMSF) and pepstatin did not influence shear force or protein degradation measured 8 d postmortem, confirming that neither serine nor aspartic proteinases affect tenderization. Injection of leupeptin, an epoxysuccinyl peptide (E-64), or N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I) blocked tenderization completely, as observed by higher (P < .05) shear force values. A causal relationship between increased toughness and prevented action of the cysteine proteinases was suggested by a concomitant reduction of myofibrillar protein degradation, generally reflected in higher (P < .05) remaining troponin-T and titin amounts and lower (P < .05) levels of 30-kDa peptide, as evaluated by semiquantitative SDS-PAGE. Moreover, parallel to these changes, amounts of salt-soluble myofibrillar protein and semiquantitative concentrations of individual salt-soluble proteins (SDS-PAGE) were also reduced (P < .05). Injection of Triton-X-100 and Ca2+ increased (P < .05) tenderness, as well as myofibrillar protein degradation and solubility, and free Ca2+, whereas EDTA induced the opposite results, indicating an important role for calpains in tenderization. Because cathepsin B, D, H, and L inhibitors did not affect texture or proteolysis, our results suggest that calpains are the main proteases involved in beef tenderization.

Topics: Animals; Calcimycin; Calcium; Calpain; Cathepsins; Cattle; Cysteine Proteinase Inhibitors; Diazomethane; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Glycoproteins; Leucine; Leupeptins; Male; Meat; Muscle Proteins; Muscles; Octoxynol; Pepstatins; Phenylmethylsulfonyl Fluoride; Postmortem Changes; Solubility

Endocytosis and subsequent degradation of iduronic acid-rich small dermatan sulfate proteoglycan from fibroblast secretions were studied in human fibroblasts. Upon endocytosis of [3H]leucine- and [35S]sulfate-labeled proteoglycan release of free leucine was 10 to 15 times more rapid than that of inorganic sulfate. Within approximately 3 h a steady state was approached between transport of proteoglycan to the compartment of core protein degradation and release of free leucine. No such steady state could be found with respect to the dermatan sulfate chains. In the presence of benzyloxycarbonyl-Phe-Ala-diazomethylketone or of other SH-protease inhibitors the degradation of the protein moiety of endocytosed proteoglycan was much less inhibited than the degradation of the polysaccharide chain. Benzyloxycarbonyl-Phe-Ala-diazomethylketone did not affect the degradation of dermatan sulfate chains taken up by fluid phase endocytosis and the activities of all known dermatan sulfate-degrading enzymes. Percoll gradient centrifugation indicated that also in the presence of the protease inhibitor the partially degraded proteoglycan accumulated in dense lysosomes. The isolation of intracellular dermatan sulfate peptides and molecular size determinations of endocytosed dermatan sulfate proteoglycan supported the conclusion that a critical proteolytic step is required before the dermatan sulfate chain becomes accessible to hydrolytic enzymes.

Topics: Ammonium Chloride; Biological Transport; Cells, Cultured; Chondroitin; Chondroitin Sulfate Proteoglycans; Cycloheximide; Dermatan Sulfate; Diazomethane; Endocytosis; Fibroblasts; Half-Life; Humans; Kinetics; Leucine; Leupeptins; Lysosomes; Molecular Weight; Protease Inhibitors; Proteoglycans; Sulfates

Culture medium from rat mammary gland explants was analyzed for the presence of cysteine proteinases. In addition to a putative precursor of the lysosomal enzyme cathepsin B, a cysteine proteinase with enzymatic properties similar to those reported for cathepsin L was found. Further evidence of the cathepsin L-like nature of this activity was provided by its high sensitivity towards the diazomethane inhibitors Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2 and towards leupeptin. The secreted form of cathepsin L is distinguished from the lysosomal form by its increased stability at alkaline pH and by its larger molecular size. It may thus represent an incompletely processed precursor form of the lysosomal enzyme.

Topics: Animals; Cathepsin B; Cathepsin L; Cathepsins; Culture Techniques; Cysteine Endopeptidases; Diazomethane; Endopeptidases; Female; Hydrogen-Ion Concentration; Lactation; Leupeptins; Mammary Glands, Animal; Molecular Weight; Pregnancy; Rats; Rats, Inbred Strains; Substrate Specificity

The effect of the lysosomotropic agent NH4Cl and the proteinase inhibitors leupeptin, Z-Phe-Ala-CHN2 (benzyloxycarbonylphenylalanylalanyldiazomethane) and pepstatin on the degradation of intracellular proteins in Swiss 3T3 mouse and normal human fibroblasts in both the exponential and stationary (confluent) growth phases in nutritionally complete conditions was investigated. Inhibitory effects of all four agents on degradation in both growth states were detected. The increase in proteolysis normally occurring as cells approach confluence could be completely blocked by NH4Cl, by Z-Phe-Ala-CHN2, or by pepstatin in the presence of leupeptin. These results suggest that the lysosomal system is responsible for the regulation of proteolysis at confluence and further confirm its role in 'basal' proteolysis in growing cells.

Topics: Ammonium Chloride; Animals; Cells, Cultured; Diazomethane; Fibroblasts; Humans; Leupeptins; Lysosomes; Mice; Pepstatins; Proteins