sodium-dodecyl-sulfate and chymostatin

Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45 degrees C. Gelatin-SDS-PAGE electrophoresis separated several bands (58-112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process.

Topics: Antipain; Bacterial Proteins; Cell Division; Chelating Agents; Cytosol; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Enzyme Inhibitors; Hydrogen-Ion Concentration; Iodoacetamide; Oligopeptides; Paracoccidioides; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Serine Proteinase Inhibitors; Sodium Dodecyl Sulfate; Temperature

We investigated and characterized the ATP-dependent protease in human erythroleukemia, K562 cells. The succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in a K562 lysate at pH 9 rose more than 10-fold with the addition of 1 mM ATP. The effect of ATP on the protease activity was dose-dependent and inhibited by the addition of ADP. This activity was not inhibited by EDTA, L-3-carboxy-trans-2,3-epoxypropionyl-leucylamide-(4-guanidin o)butane or leupeptin, but was strongly inhibited by chymostatin and diisopropylfluorophosphate. The protease activity was eluted just after the void volume from a G3000SW HPLC column. The above results suggest that this protease is identical to the high-molecular-mass protease, ingensin, previously reported by us. The ATP-dependent increase in the protease activity was due to prevention of the inactivation of the protease by ATP, and not to activation of the protease itself in the reaction mixture at 37 degrees C. The depressed succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide hydrolytic activity in the ATP-depleted lysate was restored to the same level by the detergent, SDS. Therefore, we conclude that the inactivation of ingensin occurring on preincubation is not irreversible.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Chromatography, Gel; Chromatography, High Pressure Liquid; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Edetic Acid; Enzyme Activation; Enzyme Reactivators; Humans; Hydrogen-Ion Concentration; Hydrolysis; Isoflurophate; Kinetics; Leukemia, Erythroblastic, Acute; Multienzyme Complexes; Oligopeptides; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Sodium Dodecyl Sulfate; Substrate Specificity; Tumor Cells, Cultured

A multicatalytic proteinase from rat skeletal muscle contains active site(s) catalysing the degradation of benzoyl-Val-Gly-Arg 4-methyl-7-coumarylamide, succinyl-Ala-Ala-Phe 4-methylcoumarylamide and [14C]methylcasein as well as benzyloxy-carbonyl-Leu-Leu-Glu 2-naphthylamide. These activities are 7-14-fold activated by 1 mM-sodium dodecyl sulphate. The activation leads to a higher susceptibility to the proteinase inhibitor chymostatin and to a lower ability to be inhibited and precipitated by antibodies raised against the non-activated enzyme. Since no changes in Mr or subunit composition were observed in the SDS-activated form, some conformational changes seem to occur during the activation step. More pronounced activation was observed in the presence of physiological concentrations of fatty acids; oleic acid at 100 microM concentrations stimulated the proteinase about 50-fold. In contrast with the non-activated proteinase, the activated enzyme considerably degrades muscle cytoplasmic proteins in vitro. Thus it is not unlikely that, in vivo, potential activators such as fatty acids can induce the multicatalytic proteinase to participate in muscle protein breakdown.

Topics: Animals; Binding Sites; Cysteine Endopeptidases; Endopeptidases; Enzyme Activation; Fatty Acids; Hydrolysis; Immunoelectrophoresis, Two-Dimensional; Multienzyme Complexes; Muscle Proteins; Muscles; Oligopeptides; Peptides; Proteasome Endopeptidase Complex; Rats; Rats, Inbred Strains; Sodium Dodecyl Sulfate