A particle-associated ATP-dependent proteolytic activity in erythroleukemia cells

An ATP-dependent proteolytic activity has been detected in both mouse erythroleukemia (Friend) cells and human (K562) erythroleukemia cells. Exposure of the Friend cells to dimethyl sulfoxide, which stimulates differentiation, increased ATP-dependent proteolysis approximately 2-fold although inducing differentiation in the K562 line had no significant effect on proteolysis. In contrast to the previously described soluble ATP-dependent proteolytic system of reticulocytes, the activity in the more primative erythroid cells is associated with a particulate fraction and is readily sedimentable by centrifugation at 100,000 X g for 1 h. Like the soluble reticulocyte system, the particulate activity requires divalent cation and is inhibited by hemin as well as vanadate. The activity was isolated on a sucrose cushion (30%) and did not appear to be associated with membranes, cytoskeleton, or polysomes. This enzymatic activity which degrades abnormal globin chains may initially reside in a particulate fraction and then become solubilized during erythroid maturation to the reticulocyte stage. Alternatively, the particulate activity may disappear with cell maturation being replaced by a distinct soluble activity. ATP-dependent proteolytic activity is eventually lost with reticulocyte maturation and further aging of erythrocytes.     

Proteolysis in homogenates of perfused rat liver: responses to insulin, glucagon and amino acids

The proteolytic release of leucine and isoleucine was assessed in homogenates of rat livers perfused under conditions known to influence protein degradation in the intact liver. Release was increased by perfusion alone and by additions of glucagon and was inhibited by insulin and amino acids. These responses correlated both with rates of proteolysis during perfusion and with physical alterations of the lysosomal system, reported earlier. Homogenate proteolysis appeared to comprise two components: the release of free amino acids from the total particulate fraction and from peptides in the cytosol. Both components are believed to be generated by elements of the lysosomal system.     

Purification and characterization of a membrane-associated cAMP-binding protein from developing Dictyostelium discoideum

Plasma membranes of 6-h differentiated Dictyostelium discoideum cells contain a cAMP-binding protein with the properties ascribed to the chemotaxis receptor present on these cells. We have purified this cAMP-binding protein using DEAE-Sephadex chromatography, hydrophobic chromatography on decylagarose and preparative polyacrylamide gel electrophoresis in nonionic detergent. Photoaffinity labeling of the DEAE-purified material with 8-azido-[32P] cAMP shows that only an Mr = 70,000 species on sodium dodecyl sulfate gels contains a cAMP-binding site. Two-dimensional polyacrylamide gel electrophoresis of material eluted from decyl-agarose and photoaffinity labeled indicates that the cAMP-binding protein is the most acidic of many Mr = 70,000 proteins present. This method is readily scaled up to process up to 10(11) cells which yield from 25 to 100 micrograms of cAMP-binding protein. Nucleotide specificity studies established that the cAMP-binding site of the protein is similar to that of the cAMP receptor assayed on intact cells and membranes. The rates of association and dissociation of the cAMP-binding protein are extremely rapid as found for the receptor, and its affinity for cAMP is comparable. The cAMP-binding protein is a concanavalin A binding glycoprotein, and is resistant to proteolysis by trypsin, but not chymotrypsin. Like the cAMP receptor in membranes and crude detergent extracts, this cAMP-binding protein is inhibited by phenylmethylsulfonyl fluoride. The purified binding protein exists in solution largely as a monomeric species, with some dimer being detected on gel filtration. Based on these criteria, we conclude that this cAMP binding protein represents the binding subunit of the cAMP chemotaxis receptor.     

Expressions of the c-Ha-ras and c-myc genes in rat liver tumors

Expressions of the c-Ha-ras and c-myc genes were studied by Northern blotting of total RNA from primary tumors and non-tumorous parts of the liver of rats given diet containing 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and from established rat hepatoma cell lines. The expression of the c-Ha-ras gene was found to be high in the primary tumors, non-tumorous parts of 3'-Me-DAB-treated livers and hepatoma cell lines. In contrast, the c-myc gene was expressed at a high level only in primary tumors and hepatoma cell lines. During 3'-Me-DAB treatment, the c-Ha-ras mRNA level in the liver increased by day 5 and then remained high. Increase in expression of the c-Ha-ras gene in regenerating liver was confirmed. These findings suggest that increase in expression of the c-Ha-ras gene is related to proliferation of hepatocytes, whereas expression of the c-myc gene is associated with hepatocarcinogenesis.     

Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro

Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.     

Ribozymes designed to inhibit transformation of NIH3T3 cells by the activated c-Ha-ras gene.

We have designed hammerhead ribozymes that cleave c-Ha-ras mRNA mutated at codon 12 (GGU----GUU). Plasmids containing the ribozyme-encoding genes were expressed under the control of the long terminal repeats of Rous sarcoma virus in NIH3T3 cells transfected with the activated c-Ha-ras gene. These ribozymes were found to inhibit formation of foci (by about 50%) by cleaving the oncogene mRNA, rather than by hybridizing to it. Furthermore, when the activated c-Ha-ras gene was cotransfected with the ribozyme-encoding gene, three morphologically flat colonies were found and isolated. We also found that expression of c-Ha-ras was suppressed in cells containing ribozymes.     

Inhibition of cathepsin L-induced degradation of epidermal growth factor receptors by c-Ha-ras gene products

The inhibitory activities of c-Ha-ras gene products (p21s) toward several cysteine proteinases have been investigated. The activity of cathepsin L was inhibited by p21s most effectively while those of cathepsin B and papain were slightly inhibited by p21s. p21s did not show any inhibitory activity toward cathepsin H. In order to connect the protease-inhibitor activity of p21s with cell growth, the degradation of epidermal growth factor receptors (EGF-receptors) was investigated. EGF-receptors were preferentially cleaved by cathepsin L but not by cathepsin B or H. The cleavage of EGF-receptors by cathepsin L was inhibited by p21s dose-dependently. These results raise the possibility that p21s can suppress the degradation of growth-related proteins such as EGF-receptors and thereby affect cell growth.     

Effect of pH on protease activities in the large intestine

The influence of pH on proteolysis in different fractions of human faeces was studied with a variety of chromogenic substrates. The pH optima of proteases in the washed cell fraction and washed particulate fraction were neutral to alkaline, whereas extra-cellular proteolysis in the cell-free supernatant fraction was relatively insensitive to pH over the range 6·4–8·0. Measurements with p -nitroanilide substrates suggested the presence of more than one elastase-like, trypsin-like and chymotrypsin-like protease in the gut.     

Lipase-induced alterations of fatty acid synthesis by subcellular fractions from germinating pea (Pisum sativum L.).

1. The effect of exogenous lipases on fatty acid synthesis from [14C]malonyl-CoA by the microsomal and soluble fractions from germinating peas was studied. 2. Addition of phospholipase A2 or the lipase from Rhizopus arrhizus had no effect on total fatty acid synthesis by the soluble fraction but caused severe inhibition of that by the microsomal fraction. 3. The addition of enzymes with phospholipase activity particularly inhibited the microsomal stearate elongase. 4. Control studies indicated that the phospholipase-induced inhibition of fatty acid synthesis was due to the location of fatty acid synthetase, palmitate elongase and stearate elongase on the outside of the microsomal vesicles. 5. Experiments with a trypsin-like proteinase showed that approximately half the microsomal fatty acid synthesis was resistant to proteolysis. 6. Although addition of exogenous phospholipases had no effect on total fatty acid synthesis by the soluble fraction, it did increase alpha-hydroxylation of newly-formed palmitate and stearate. 7. The results provide further evidence for differences between the soluble and particulate fatty acid synthetase and palmitate elongase activities of germinating pea.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p-nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis-type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Contribution of the microflora to proteolysis in the human large intestine

Protease activities in human ileal effluent were approximately 20-fold greater than in normal faeces. Comparative studies with faeces from a person who did not have a pancreas suggested that a substantial proportion of the proteolytic activity in normal faeces was of bacterial origin. Thimerosal, iodoacetate, EDTA and cysteine significantly inhibited proteolysis in faeces, but not in small intestinal contents, showing that cysteine and metalloproteases were produced by bacteria in the large gut. These results, together with results from studies using p -nitroanilide substrates, demonstrated that faecal proteolysis was both qualitatively and quantitatively different from that in the small intestine. Studies with pure cultures of proteolytic gut bacteria indicated that the cell-bound proteases of Bacteroides fragilis -type organisms were likely to contribute significantly towards proteolytic activity associated with the washed cell fraction and washed particulate fraction of faeces. Extracellular proteases were formed by Streptococcus faecalis ST6, Propionibacterium acnes P6, Clostridium perfringens C16, Cl. bifermentans C21 and Cl. sporogenes C25. Inhibition results suggested that these bacteria, and similar organisms, may be partly responsible for the extracellular proteolytic activity found in the cell-free supernatant fraction of faeces.     

Endocytosis, proteolysis, and exocytosis of exogenous proteins by cultured myotubes

The endocytic activity of chick myotubes in cultures was investigated. Differentiated myotubes internalized the fluid-phase marker horseradish peroxidase in membrane-bound particles which typically displayed reaction product at the inner surface of the vesicle. Pulse-chase experiments demonstrated a rapid decrease in the number of horseradish peroxidase-containing vesicles and a redistribution from a uniform to a perinuclear pattern. Horseradish peroxidase uptake was extensively inhibited by incubation at 0-4 degrees C consistent with an endocytic mechanism. To further characterize the process, the fate of labeled protein was investigated. Following uptake [3H] hemoglobin A was extensively degraded (40-50%) to acid-soluble products within 10 h. Degradation displayed a biphasic pattern with a rapid early phase followed by a much slower second phase. The decreasing rate of proteolysis can be accounted for, in part, by a simultaneous exocytosis of a substantial fraction (25-30%) of acid-insoluble label from myotubes. The lysosomotropic agents methylamine, monensin, and chloroquine significantly inhibited (23-75%) proteolysis, indicating a lysosomal site of degradation. Part of the inhibitory effect results from an increase in exocytosis in the presence of these agents. Degradation of endocytosed [3H]hemoglobin A was not inhibited by insulin. In contrast degradation of endogenous myotube proteins was inhibited (40%) by insulin and blocked by methylamine. These results suggest that cultured myotubes possess a coupled endocytic/exocytic pathway for macromolecules and that a fraction of the internalized substrate is degraded by an insulin-insensitive lysosomal pathway.     

Peroxidatic degradation and ether link cleavage of thyroxine in a particulate fraction of human thyroid

The present study was undertaken to investigate degradation of thyroxine (T4) mediated by thyroid peroxidase in man. A particulate fraction (1,000-100,000 x g) of normal human thyroid tissue was prepared and used as crude enzyme. 125I-T4 and unlabeled T4 were incubated with the particulate fraction in buffer containing glucose and glucose oxidase for generation of H2O2. After incubation, iodoamino acids were extracted with ethanol and the products of T4 degradation were analyzed by thin layer chromatography. In this system, T4 was degraded in time-, temperature- and pH-dependent manners, but not in the absence of the H2O2-generating system. The rate of degradation was related to concentration of the particulate fraction. The reaction was inhibited by methimazole, propylthiouracil and catalase. When [3',5'-125I] T4 was used as a tracer, major labeled products of T4 degradation were inorganic iodide and ethanol-unextracted fraction and no detectable labeled 3,5,3'-triiodothyronine (T3) or 3,3',5'-triiodothyronine (rT3) was generated. From a kinetic study by adding various doses of unlabeled T4, the apparent Km value for T4 was 30 microM and the Vmax value was 230 pmol/mg protein/min. When [3,5-125I] T4 was incubated with enzyme preparation, one third of degraded T4 was recovered as diiodotyrosine (DIT) and half of 125I-DIT was degraded in parallel incubation. No formation of radiolabeled DIT was observed in incubation with Na- 125I done in tandem. These findings suggest that thyroid hormones can be metabolized by peroxidase in human thyroid by pathways that include cleavage of ether linkage.     

Proteinase Enzyme System of Lactic Streptococci: I. Isolation and Partial Characterization1

Proteinase activity of Streptococcus lactis cells was maximal at pH 6.0, but after storage at 3 C the minimal activity was observed at this pH. Activity lost as a result of storage could be restored by adding glutathione. Whole cells were fractionated into soluble (intracellular) and particulate fractions by sonic disruption; both fractions contained enzymatic activity. Activity of the soluble (intracellular) fraction was found to be stable to storage at 3 C, but was inhibited progressively with increasing concentrations of p-hydroxymercuribenzoate (PHMB). The enzymatic activity of this fraction was not activated by ferrous or magnesium ions or by cysteine. In contrast, activity of the particulate fraction was labile to storage at 3 C, and the reduction was comparable to that of stored cells. Furthermore, proteinase activity in the cells and the particulate fraction was not affected by addition of PHMB. The particulate fraction was activated by ferrous and magnesium ions and by cysteine. After storage, only ferrous ion and cysteine promoted reactivation; magnesium ion was totally ineffective. The enzyme(s) contained in the particulate fraction may be involved in decreased proteinase activity observed in whole cells and in the effect on growth of cells after storage.     

Proteolysis of canine apolipoprotein by acid proteases in canine liver lysosomes.

Canine liver lysosomes were purified by sucrose discontinuous density gradient centrifugation and then ruptured by sonication to obtain the soluble fraction. This soluble lysosomal fraction, which contained a 25-fold increase in acid phosphatase activity per mg of total protein when compared with the original homogenate, was incubated with a subfraction (1.110 less than d less than 1.210 g/cm3, HDL3) of canine high density lipoproteins (HDL) at pH 3.8. HDL3 proteolysis by lysosomal proteases, measured as the release of peptides and amino acids by the ninhydrin reaction, followed hyperbolic curves with straight lines (r = 0.99) obtained on Lineweaver-Burk plots. Km calculated from the Lineweaver-Burk plot was 635 mug of HDL3 protein per 0.5 ml of incubation mixture. Optimum HDL3 proteolysis was observed from pH 3.8 to 4.5. Incubation with the other subcellular organelle fractions did not result in HDL3 proteolysis. To evaluate the effects of enzyme inhibitors, iodoacetate, p-chloromercuribenzoate (both specific for the endopeptidase, cathepsin B (EC 3.4.22.1)) and pepstatin (specific for the endopeptidase, cathepsin D (EC 3.4.23.5) were tested. Iodoacetate and p-chloromercuribenzoate inhibited HDL3 proteolysis 100% and bovine serum albumin proteolysis 65%. Pepstatin inhibited HDL3 proteolysis 45% and bovine serum albumin proteolysis 70%. The in vitro data presented support the hypothesis that hepatic lysosomes play an important role in HDL3 catabolism in the dog. Furthermore, results obtained from enzyme inhibition studies suggest that a specific lysosomal endopeptidase, cathepsin B, may play the key role in HDL3 proteolysis.     

Mutations induced by DNA lesions in hot spots of the c-Ha-ras gene.

In order to investigate whether several DNA lesions (O6-methylguanine, 8-hydroxyguanine, xanthine, an abasic site analogue and hypoxanthine) activate a c-Ha-ras gene and to determine the type of mutations induced by the DNA lesions, they were introduced into a synthetic c-Ha-ras gene by DNA cassette mutagenesis techniques. The modified genes were transfected into mouse NIH3T3 cells and the c-Ha-ras genes present in transformed cells were analysed. O6-methylguanine and xanthine induced a mutation to A, hypoxanthine induced a mutation to G. 8-hydroxyguanine and the abasic site analogue caused random mutations in the modified and adjacent positions. These results indicated that the synthetic c-Ha-ras gene is very useful for the detection of mutations caused by a DNA lesion.     

Insulin differentially regulates protein phosphotyrosine phosphatase activity in rat hepatoma cells.

We have studied the effect of insulin stimulation on phosphotyrosine phosphatase (PTPase) activity in the well-differentiated rat hepatoma cell line Fao. PTPase activity was measured using a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation. Of the PTPase activity in Fao cells, 14% was in the cytosolic fraction, whereas 86% was in the particulate fraction; this latter fraction also had a 4-fold higher specific activity. Purification of the particulate fraction by lectin chromatography resulted in a 50% increase in specific activity, although this glycoprotein-rich fraction contained only 1.5% of the total activity. Both the cytosolic and particulate PTPase fractions were active toward the tyrosyl-phosphorylated insulin receptor in vitro. The activity of the particulate fraction but not the cytosolic fraction was inhibited by addition of a micromolar concentration of a phosphorylated peptide corresponding to residues 1142-1153 of the human insulin receptor sequence. By contrast, addition of the nonphosphorylated peptide even at millimolar concentration was without effect. Both PTPase fractions were inhibited by Zn+ at similar concentrations, whereas the cytosolic PTPase activity was 10-fold more sensitive to vanadate inhibition. Treatment of cells with 100 nM insulin increased PTPase activity in the particulate fraction by 40% and decreased activity in the cytosolic fraction by 35%. These effects occurred within 15 min and were half-maximal at 3-4 nM insulin. When assessed as total activity, the magnitude of the changes in PTPase activity in the particulate and cytosolic fractions could not be explained on the basis of a translocation of PTPases between the two pools.(ABSTRACT TRUNCATED AT 250 WORDS)