Inactivation and degradation of rat liver catalase by mitochondrial and lysosomal proteinases

The initial phases of catalase degradation in rat hepatocytes were studied. Preparations of highly purified fractions of lysosomes and mitochondria from rat liver were obtained. The proteinase activity was measured by the radio-isotope method by the increase of the free amino groups or by the decrease of the catalase activity, using labelled catalase as a substrate. It was found that the initial step of catalase degradation occurs in the enzyme localized in the inner membrane as well as in the mitochondrial matrix and that the total degradation of catalase is completed in the lysosomal fraction of rat liver.     

Purification and properties of recombinant rat catalase produced in Escherichia coli

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.     

Neutral proteinase in peroxisomes from rat liver cells

A proteinase with a pH optimum of 7.6 and Mr of 43 kD has been isolated from rat liver peroxisomes. The peroxisomes were shown to possess an intrinsic mechanism responsible for the degradation of proteins, e.g., catalase. Some physico-chemical properties and turnover parameters of neutral proteinase from peroxisomes (i.e., synthesis rate, lifetime, degradation of the newly synthesized enzyme) were studied. In terms of enzymatic and immunologic properties, the enzyme under study is similar to mitochondrial and cytosolic proteinases responsible for the initial inactivation of catalase in subcellular structures.     

Purification and characterization of a rat liver ferroactivator with catalase activity

A rat liver protein with both phosphoenolpyruvate carboxykinase ferroactivator activity and catalase activity has been purified to near-homogeneity. The protein has a native molecular weight of 240,000 and is composed of four identical subunits containing ferriprotoporphyrin IX prosthetic groups. The visible spectrum has absorbance maxima at 403, 500, 530, and 620 nm; it is not reduced by dithionite. The spectrum, physical properties, and specific activity are almost identical with those of catalases from other sources, and the protein has been tentatively identified as rat liver catalase. The protein exhibited partial reactivity in double immunodiffusion plates to antiserum prepared against rat liver ferroactivator isolated by a previous method (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440) raising the possibility that the original ferroactivator and rat liver catalase are structurally related. Inactivation of catalase by 3-amino-1,2,4-triazole was accompanied by loss of ferroactivator activity as well. The apparent specific activity of ferroactivator, as well. The apparent specific activity of ferroactivator, whether heme-containing or not, can be increased between 2- and 100-fold by the inclusion of bovine serum albumin, HCO3-, or a combination of the two in the incubation.     

Amino acid sequence of rat epidermal thiol proteinase inhibitor

The complete amino acid sequence of rat epidermal thiol proteinase inhibitor was determined. The unique 103-residue sequence was derived by analysis of two peptides generated by limited proteolysis of the native inhibitor with Staphylococcus aureus V8 protease and of three cyanogen bromide fragments. The protein has a high degree of sequence homology to either rat liver or human leucocyte inhibitor but is not identical and may represent a new type of low molecular weight thiol proteinase inhibitors.     

Membrane proteinases from normal and neoplastic tissues in man and the rat

Plasma membranes were purified from rat liver, muscle and sarcoma tissues and from human liver and hepatoma tissues. The plasma membranes all contained DFP-sensitive, neutral proteolytic activity. Plasma membranes from all normal tissues contained a single DFP-binding protein of apparent molecular weight 68,000. Only the plasma membranes from tumour tissue contained a plasminogen activator; the DFP-binding proteins from these membranes were more diverse than those from the normal samples. The rat liver plasma membrane proteinase was purified. It was a labile enzyme sensitive to inhibition by DFP and by calcium ions, and with a broad substrate specificity. A similar protein was the sole DFP-binding protein in rat liver microsomes. This and the properties of the enzyme suggested a possible role in the processing and secretion of newly-synthesized protein.     

Comparison of properties of thiol proteinase inhibitors from rat serum and liver

Thiol proteinase inhibitors in rat serum were purified and their properties were compared with those of rat liver thiol proteinase inhibitor. The inhibitors in rat serum were separated into three forms (S-1, S-2, and S-3) by linear gradient elution from a DE52 column. One inhibitor (S1) was purified to homogeneity by chromatography on ficin-bound Sepharose and Sephadex G-150 columns. The apparent molecular weights of S1, S2, and S3 on Sephadex G-150 columns were 90,000, 95,000, and 160,000, respectively. Serum thiol proteinase inhibitor and liver thiol proteinase differed in the following: 1) all three forms of serum inhibitor had much higher molecular weights than the liver thiol proteinase inhibitor (Mr = 12,500); 2) no cross-reactivity was observed between serum inhibitors and liver inhibitor in tests with either antiserum inhibitor or anti-liver antiserum; 3) both serum inhibitor and liver inhibitor were specific for thiol proteinases, but had different inhibition spectra; 4) the liver inhibitor did not bind to concanavalin A-Sepharose, whereas the serum inhibitor bound and was eluted with alpha-methyl mannoside. A thiol proteinase inhibitor of high molecular weight detected in tissue homogenates inhibited papain markedly but did not inhibit cathepsin H. Its activity was diminished by perfusion of the organ, indicating that it is derived from serum.     

A 25 000 dalton inhibitor of cAMP independent protein kinases present in rat liver HMG protein preparations

A protein kinase inhibitor was found in rat liver cells as a component of HMG proteins. It is located in cytosol as well as in nuclei. It inhibits all tested cAMP independent protein kinases and has no effect on cAMP dependent protein kinases. This inhibitor is a 25 000 Da protein. It has no ATPase, phosphoprotein phosphatase or proteinase activity and is heat unstable.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

大鼠肝脏过氧化氢酶的分离纯化及性质

根据过氧化氢酶的催化特性,采用盐析,透析,离心等技术,从大鼠肝脏中分离纯化过氧化氢酶,提纯倍数达到26倍,酶活性回收率为57%。为一般实验室分离纯化大鼠过氧化氢酶提供了一种有效的方法。酶学性质研究表明,该酶最适温度为37℃,最适pH值为7.5,在此条件下,以过氧化氢为底物的Km值为56 mmol.L-1。     

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59 789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC posses an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE. Mol. Reprod. Dev. 51:265–273, 1998. © 1998 Wiley-Liss, Inc.     

Detection of catalase in rat heart mitochondria.

The presence of heme-containing catalase in rat heart mitochondria (20 +/- 5 units/mg) was demonstrated by biochemical and immunocytochemical analysis. Intact rat heart mitochondria efficiently consumed exogenously added H2O2. The rate of H2O2 consumption was not influenced by succinate, glutamate/malate, or N-ethylmaleimide but was significantly inhibited by cyanide. Hydrogen peroxide decomposition by mitochondria yielded molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism. Mitochondrial fractionation studies and quantitative electron microscopic immunocytochemistry revealed that most catalase was matrix-associated. Electrophoretic analysis and Western blotting of the mitochondrial matrix fraction indicated the presence of a protein with similar electrophoretic mobility to bovine and rat liver catalase and immunoreactive to anti-catalase antibody. Myocardial tissue has a lower catalase-specific activity and a greater mitochondrial H2O2 production/g of tissue than most organs. Thus catalase, representing 0.025% of heart mitochondrial protein, is important for detoxifying mitochondrial derived H2O2 and represents a key antioxidant defense mechanism for myocardial tissue.     

Electron microscopic localization of the multicatalytic proteinase complex in rat liver and in cultured cells.

The multicatalytic proteinase (MCP) prosome or proteasome is a large multifunctional complex which is believed to play a major role in non-lysosomal pathways of intracellular protein degradation and has recently been implicated in antigen processing. In this study, affinity-purified antibodies against rat liver MCP were used to investigate the localization of the proteinase both in rat liver and in growing human L-132 cells in culture, using electron microscopic immunogold techniques. Quantitation of the MCP in different subcellular localizations by morphometric analysis of electron micrographs showed the proportion in the nucleus to be 17% for hepatocytes and 51% for L-132 cells, demonstrating differences in the distribution of MCP in different cell types. In hepatocytes, 14% of the total MCP was found associated with the endoplasmic reticulum. The remainder was localized in the cytoplasmic matrix. Immunofluorescence studies with L-132 cells also showed a reaction in nuclei and cytoplasm. The localization of MCP is consistent with its proposed multiple functions in protein turnover, in the production of peptides for antigen presentation, and in RNA processing.     

Changes in the nuclear proteinase activity of the liver in gamma-irradiated rats

Activity of nuclear proteinases in rat liver was studied, by their capacity of splitting a casein substrate, 5-48 h following gamma irradiation. The proteinase activity of nuclei was shown to increase by more than two times in 2-5 h and to decrease by 3-4 times after 15-48 h compared to that of the nonirradiated controls. A sharp decrease in the proteinase activity was also noted in the nuclear matrix of irradiated rat liver.     

Purification and characterization of thiol proteinase inhibitor from rat liver

A thiol proteinase inhibitor was purified from rat liver by essentially the same procedure as reported previously (Kominami, E., Wakamatsu, N., and Katunuma, N. (1981) Biochem. Biophys. Res. Commun. 99, 568-575), but without heat treatment. The purified inhibitor appears homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor has Mr = 12,500 and contains 50.5% of polar amino acid residues, 9.3% aromatic amino acids, and no tryptophan. The presence of 2 half-cystines/molecule and the absence of free thiol groups indicate that the inhibitor possesses one disulfide bridges. The inhibitor inhibits cathepsin H by forming an enzyme-inhibitor complex in a molar ratio of 1:1. It inhibits most thiol proteinases such as cathepsin H, L, B, and C, papain, and ficin, but not calcium-activated neutral proteinase or serine proteinases or carboxyl proteinases. The inhibitor was found in various rat tissues. Immunological diffusion analysis with anti-liver thiol proteinase inhibitor serum indicated that the rat liver inhibitor is immunologically identical with the inhibitors from other rat tissues. On subcellular fractionation of rat liver, the thiol proteinase inhibitor was recovered in the cytosol fraction.     

Catalase takes part in rat liver mitochondria oxidative stress defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism with a rate constant of 0.0346 s(-1). The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 +/- 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.     

Changes in cell membrane proteins during N-nitrosodiethylamine induced carcinogenesis

The composition of rat liver cell membrane proteins during the N-nitrosodiethylamine-induced hepatocarcinogenesis was studied using polyacrylamide gel electrophoresis. The effect of the carcinogen on rat liver was controlled by the catalase activity in the microsomal fractions. The plasma membrane protein fractions with Mr of 140 kDa, 135 kDa, 40 kDa and 22 kDa as well as the 36 kDa fraction from endoplasmic reticulum membranes were found to decrease during hepatocarcinogenesis.     

Electron paramagnetic resonance spectra of catalase in mammalian tissues.

The relatively small number of paramagnetic species and the high concentration of catalase in mammalian liver and blood make it possible to directly study this enzyme in frozen whole tissue. The EPR spectra of catalase are dependent on the heme environment and in human blood only catalase A, gxy = 6.48, 5.36 is observed whereas in liver a second spectrum, catalase B, gxy = 6.80, 5.07 can also be seen. Using rapid freeze techniques it has been shown that in rat liver catalase A corresponds to the in vivo steady state and that after death this is largely converted into catalase B. Data from the perfusion of rat livers with oxygenated and deoxygenated blood and dextran solutions together with results from in vitro studies of catalase are interpreted as indicating that catalase B results from the interaction of catalase with an organic acid, most probably formic acid, that the acid is a peroxidative substrate for catalase in vivo and that peroxidation of the acid is not the major role for catalase in rat liver. Catalase binding with other small molecules in intact liver has been demonstrated by perfusion with nitrite-containing dextrans and by intraperitoneal injection of 3-amino-1,2,4-triazole.     

Metabolism and various properties of proteinase controlling catalase metabolism in rat liver mitochondria

The Mg,ATP-dependent serine proteinase (Mr = 50 kD; pH optimum 8.0) was isolated and purified 750-fold. The substrate specificity of the enzyme to some protein substrates (catalase, aldolase, uratoxidase, superoxide dismutase, albumin, cytochrome c, insulin) was investigated. The proteinase shows an affinity for proteins whose molecular mass is more than 100 kD. Some quantitative parameters of the enzyme metabolism, e.g., rate constants for synthesis and degradation of serine proteinase and the time of functioning of the de novo synthesized protein, were investigated.