Enzyme & protein最新文献

Synthetic inhibitors of the multicatalytic proteinase complex (proteasome) can provide the means to uncover the functional significance and catalytic mechanism of this macromolecule. Although inhibitor development is still in its early stages, some useful compounds have already been prepared. Of the various types of inhibitors thus far studied, peptidyl aldehydes have been the most effective. Since peptidyl aldehydes inhibit both serine and cysteine proteinases, lack of specificity is their major limitation. The properties of one such compound N-benzyloxycarbonyl-IE(Ot-Bu)A-Leucinal, a potent inhibitor of suc-LLVY-MCA hydrolysis, are described in detail.

Proteasomes are large multicatalytic protease complexes found in the cytoplasm and nucleus of all eukaryotic cells. 20S proteasomes are cylindrically shaped particles composed of a set of different subunits arranged in a stack of 4 rings with 7-fold symmetry. In yeast 14 different genes are known, which are proposed to code for the complete set of 20S proteasomal subunits. They can be divided in 7 alpha- and 7 beta-type subunits. 26S proteasomes are even larger proteinase complexes which contain the 20S proteasome as the functional proteolytic core. They degrade ubiquitinylated proteins in vitro. Several yeast 26S proteasome subunits have been characterized as members of a novel ATPase family. Studies with yeast 20S and 26S proteasome mutants uncovered the function of proteasomes in stress-dependent and ubiquitin-mediated proteolytic pathways. Proteasomes are important for cellular regulation, cell differentiation, adaptation to environmental changes and are involved in cell cycle control.

Subunits from human placental proteasomes were separated by two-dimensional polyacrylamide gel electrophoresis. The amino acid composition of proteins from individual spots were determined. Some of the spots had identical amino acid compositions, confirming that they contain isoforms of the same subunit. Proteasomes from HeLa cells, labelled with 3H-leucine, were precipitated with an antibody and similarly separated into subunits. The radioactivity in each subunit was measured. The subunit stoichiometry was then calculated from these data and the leucine contents in the subunits. Each of the 14 major subunits of human proteasomes are apparently present in equal amounts.

In studying the modulation of multicatalytic proteinase (MCP), we have focused on three main aspects: (1) modulation of the activity of the MCP complex by lipids, showing that cardiolipin, sulfatides and gangliosides are potent activators of the enzymatic activity of the complex; (2) modulation by interconversion of MCP subunits, showing that casein kinase II is able to phosphorylate the C8 (this subunit is also be main in vivo phosphorylated subunit) and C9 subunits of the complex in vitro and that a 26-kD subunit is phosphorylated in vitro by protein kinase C, and (3) modulation by proteolytic processing, extending our previous observation of proteolytic processing of the C2 COOH terminus, the presence of enzymatic activities in different subcellular fractions able to convert the intact C2 (32 kD) subunit to a 28-kD polypeptide by removal of at least the last 9-13 amino acids of the C2 polypeptide. The data presented illustrate that the MCP complex is probably under tight and multifactorial control in vivo.

A sensitive radiochemical method for the determination of the pyruvate dehydrogenase complex (PDHC) activity in skeletal muscle tissue, based on the decarboxylation of [1-14C]-pyruvate to 14CO2, is described. Measurements can be carried out either in muscle homogenate or in 600-g supernatant, both obtainable from a small muscle biopsy specimen (20 mg). In addition to NAD+, thiamine pyrophosphate and coenzyme A in the incubation mixture, a preparation of NADH:cytochrome c reductase (NADHCR) together with cytochrome c has a stimulating effect on the PDHC activity. NADHCR constitutes an oxidation system for NADH to prevent feedback inhibition. Addition of L-carnitine also results in stimulation of PDHC by trapping the produced acetyl-CoA as acetylcarnitine. Special care for radioactive pyruvate, with freeze drying and storage at -20 degrees C under nitrogen, and determination of the purity during every PDHC assay, is required. In the presented assay a Km value of 0.084 mmol/l was found for pyruvate. Nonsigmoidal kinetics was found with a Hill coefficient of 1.63. With the described method, a totally Mg2+,Ca(2+)-stimulated PDHC activity is measured. Addition of a purified specific pyruvate dehydrogenase phosphatase did not yield a higher PDHC activity. Finally, comparison of total PDHC activity with [1-14C]-pyruvate oxidation rates, both measured in the supernatant prepared from fresh muscle, shows an equimolar correlation, indicating that total PDHC activity is rate limiting in the assay for the pyruvate oxidation rate. Neonatal muscle exhibits five to ten times lower PDHC activities and pyruvate oxidation rates than controls (age > 3 years).

Little is known about the kinetics of most serum enzymes during the first hours of life, and even less about the effect on such enzyme activities of perinatal hypoxia-ischaemia. It was the aim of the present study to evaluate the serum kinetics of seven differently located cell enzymes in healthy and asphyxiated newborns during the 1st week of life. The serum activities of cytoplasmic and mitochondrial [aspartate aminotransferase (ASAT), creatine kinase (CK), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), and hydroxybutyrate dehydrogenase (HBDH)] and membrane-bound (gamma-glutamyl-transferase and leucine arylaminidase) enzymes were prospectively measured in full-term asphyxiated (n = 49) and healthy (n = 87) newborns during the first 144 h of life. The blood samples were taken serially at five fixed times: 0 (cord), 12, 24, 72, and 144 h postpartum. The asphyxiated newborns had significantly increased serum activities of ASAT, LDH, and HBDH up to 72 h postpartum, whereas healthy newborns showed higher CK and GLDH activities. Only the activities of ASAT, LDH, and HBDH seemed to depend on the oxygen supply of the fetus or newborn. If other causes of increased serum enzyme activities, e.g. liver diseases, haemolytic disorders, tumours, or inborn errors of metabolism, are excluded, elevated serum activities of ASAT, LDH, and HBDH should draw one's attention to a perinatal hypoxic-ischaemic insult of the newborn.

Two low molecular weight fibrinolytic/hemorrhagic enzymes, jararafibrase III and jararafibrase IV, were purified from Bothrops jararaca venom using a fast protein liquid chromatography system. The purified jararafibrase III and jararafibrase IV were single chain proteins with molecular weights of 20,400 +/- 500 and 21,200 +/- 400, respectively, by SDS-PAGE. The isoelectric points of jararafibrase III and jararafibrase IV were 9.4 and 6.9, respectively. The activity of the enzyme was inhibited by 1,10-phenanthroline and EDTA, suggesting that both enzymes were metalloproteinases. The specific fibrinolytic activities of jararafibrase III and jararafibrase IV were 7.5 +/- 0.4 and 6.5 +/- 1.6 units/mg protein, respectively. The enzymes induced local hemorrhage in the skin of rats. The minimal hemorrhagic doses of jararafibrase III and IV were 31.0 and 34.0 micrograms/rat, respectively. The enzymes displayed broad substrate specificities like the previously purified jararafibrases I and II. Jararabrases III and IV degraded type-IV collagen, gelatin, laminin and fibronectin into smaller fragments. The specific activities of jararafibrase III for type-IV collagen and gelatin were 7.6 +/- 0.3 and 43 +/- 11 units/mg protein, respectively. The specific activities of jararafibrase IV for type IV-collagen and gelatin were 16.5 +/- 1.2 and 112 +/- 9 units/mg protein, respectively.

We have isolated and characterized a cDNA encoding the mouse proteasome subunit MC3 and identified four proteasome subtypes which differ in their peptide-hydrolyzing and polypeptide-cleavage properties. Immunoblotting data show that the 25-kD MC3 subunit is a constitutive proteasome subunit which exists in several isoforms. In addition, by immunoprecipitation of proteasomes with AbMC3, a subset of enzyme complexes could be recognized which differ in their relative subunit composition from the bulk of proteasomes. Using DEAE-column chromatography we identified three different proteasome subtypes in sol-80 mouse liver extracts and, by Trition X-100 extraction, a distinct membrane-bound subtype. The four proteasome subtypes are shown to differ in their trypsin- and chymotrypsin-like hydrolyzing activities as well as in their ability to cleave a 25mer polypeptide substrate derived from the MCMV IE pp89. Our data indicate that the enzymatic properties observed for the total proteasome population may be the summary of cleavage properties of different types of proteasome complexes.

Alkaline phosphatase solubilized from a human Hodgkin's lymphoma cell line (L428) was compared with purified amphiphilic and hydrophilic forms of the enzyme from human liver, and with the enzyme solubilized from a cultured osteosarcoma cell line (Saos-2). Purified hydrophilic alkaline phosphatases from human placenta and intestine were also compared in some experiments. Alkaline phosphatase was released from the plasma membrane of intact lymphocytes by phosphatidylinositol phospholipase C and thus is anchored to the outside of the plasma membrane by covalently attached phosphatidylinositol. Enzyme released in this way was hydrophilic and that solubilized with Triton X-100 was amphiphilic, as assessed by adsorption to octyl-Sepharose. Lymphocyte alkaline phosphatase, when released from the membrane by phosphatidylinositol phospholipase C or solubilized by Triton X-100, had apparent M(r) values on gradient gel electrophoresis of 227 and 494 kDa, respectively. These values were consistently higher than equivalent ones obtained with enzymes purified from human liver, but were similar to those of cultured osteosarcoma cells. Isoenzyme-specific inhibitors of alkaline phosphatase showed similar patterns of inhibition between the enzyme from L428 cells and the tissue-nonspecific (liver/kidney/bone) isoenzyme from human liver. Heat stabilities were similar for the enzymes from L428 and Saos-2 (bone isoform) cell lines, but differed significantly from those of liver, intestine and placenta. We conclude that the alkaline phosphatase expressed in this lymphoma cell line (L428) has properties that most closely resemble those of the tissue-nonspecific isoenzyme found normally in osteoblasts of bone (bone isoform).