Purification and some properties of rat liver tyrosyl-tRNA synthetase.

Rat liver cytoplasmic tyrosine:tRNA ligase (tyrosine:tRNA ligase, EC 6.1.1.1) was purified by ultracentrifugation, DEAE-cellulose chromatography and repeated phosphocellulose chromatography by more than 1500-fold. The molecular weight of the enzyme was approx. 150 000 as determined by Sephadex G-200 gel filtration. On the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme consisted of two subunits, each of 68 000 daltons. We found the following Km values for the enzyme: 13 micrometer for tyrosine and 1.7 mM for ATP in the ATP:PPi exchange reaction and 13 micrometer for tyrosine, 210 micrometer for ATP and 0.14 micrometer for tRNATyr in the aminoacylation reaction. The rate of tyrosyl-tRNA synthesis was 50-fold lower than that of ATP:PPi exchange. Addition of a saturating amount of tRNA did not affect the rate of ATP:PPi exchange.     

Purification and some properties of a monoacylglycerol-hydrolyzing enzyme of rat adipose tissue.

A monoacylglycerol-hydrolyzing enzyme has been purified 2500-fold from rat adipose tissue. The key step was the solubilization of the enzyme, presumably as an enzyme-detergent complex, by sonication with a nonionic polyoxyethylene alcohol detergent. The purification was achieved by ion exchange and gel chromatography, and isoelectric focusing, in the presence of detergent. By sodium dodecyl sulfate gel electrophoresis the enzyme protein was more than 85% pure. This method indicated a minimum molecular weight of 32,900. The preliminary amino acid composition, excluding tryptophan, could best be fitted with a value of 31,800. The purified enzyme had a pI of 7.2, an estimated Stokes radius of 39 A by gel chromatography and a pH optimum of 8.0. Enzyme stability was highly dependent on presence of detergent and free sulfhydryl groups. The enzyme was responsible for the main monoacylglycerol- but only a small part of the p-nitrophenylacetate-hydrolyzing activity of crude adipose tissue extracts and hydrolyzed 1(3)- and 2-monooleoylglycerol at equal rates. Under the assay conditions used it did not catalyze the hydrolysis of emulsified trioleoylglycerol, micellar or emulsified dioleoylglycerol, emulsified cholesterol oleate or micellar lysophosphatidylcholine. It is possible that the enzyme may be a specific monoacylglycerol hydrolase.     

Purification and some properties of carboxylesterase of rat adipose tissue

Carboxyl ester hydrolase was obtained from rat epididymal adipose tissue in an electrophoretically homogeneous form. Purification was achieved by acetone precipitation, followed by successive chromatographies on DEAE-cellulose and hydroxyapatite and then isoelectric focusing. The monomeric molecular weight of the enzyme was 65 000 and the enzyme associated to form trimers. The enzyme had an isoelectric point at pH 5.9 and contained 2.1% carbohydrate moiety per protein with a molecular weight of 65 000. The amino terminal residue of the enzyme was glycine. The enzyme catalyzed the hydrolysis of short chain triacylglycerols such as tributyrin and medium chain monoacylglycerols such as monocaprin, but not the hydrolysis of cholesterol ester. The optimum pH for the enzymatic function of this enzyme for methyl butylate was 8.0. An antibody against the highly purified enzyme preparation induced in rabbits strongly inhibited the esterase of rat adipose tissue, but did not inhibit the esterase of rat liver, intestinal mucosa and serum.     

Purification and some kinetic properties of rat liver ATP citrate lyase.

A new purification procedure for rat liver ATP citrate lyase is described. The method reproducibly gives homogenous undegraded enzyme. Steady-state kinetic analysis of ATP citrate lyase was complicated by the presence of ADP, a product of the reaction, in solutions of ATP. The kinetic patterns observed were dependent on whether ADP was removed by the assay system. When assays were performed with a method in which ADP was removed, the results showed that the enzyme obeys a double-displacement mechanism with a phosphoenzyme intermediate. This resolves a controversy between the results of previous kinetic studies and those of isotope-exchange and enzyme-labelling experiments.     

A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution

Rat liver cytosol has low hydrolytic activity against [3H]methylcasein at neutrality, but activity increases greatly on addition of various compounds such as poly-L-lysine, N-ethylmaleimide, and sodium dodecyl sulfate, suggesting that it contains latent proteolytic activity. The latent enzyme was found to be stabilized in the presence of 20% glycerol and to be activated by addition of poly-L-lysine. The latent enzyme was purified from a crude extract of rat liver to apparent homogeneity in the presence of 20% glycerol by conventional chromatographic techniques. The purified enzyme showed endoproteolytic activity toward various proteins when it was activated by the compounds listed above. It preferentially degraded N-substituted tripeptide substrates with a basic amino acid at the carboxyl terminus, as well as peptides containing neutral hydrophobic amino acids. It did not require activation for these peptidase activities, in contrast to its activity toward large proteins. Interestingly, a proteinase and a trypsin-like and a chymotrypsin-like peptidase activity could not be separated by customary chromatographic methods but were distinguishable by their sensitivities to various inhibitors, activators, and covalent modifiers, suggesting that the enzyme has three distinct active sites within a single protein. The enzyme seems to be a seryl endopeptidase showing maximal activity at neutral and weakly alkaline pH values. Thus, the enzyme is a unique protease with latent multifunctional catalytic sites. The distribution of the protease in soluble extracts of various rat tissues and cells was examined quantitatively by an enzyme immunoassay. The enzyme level was highest in liver and also in spleen, stomach, lung, small intestine, and kidney, but was low in heart, diaphragm, skeletal muscle, brain, and skin. The concentrations of enzyme in some established cell lines including hepatoma and rat kidney cells were comparable to that in normal liver hepatocytes. The enzyme was found mainly in the cytosol fraction, although a small amount was associated with microsomal membranes, suggesting that it is an extralysosomal protease. Immunohistochemical staining of the liver and skeletal muscles showed that the protease is distributed diffusely in panlobular hepatocytes with slight centrilobar predominance and is present in Kupffer cells, vascular endothelial cells, and bile duct epithelial cells in the liver and also diffusely in the intermyofibrillar spaces and vascular endothelial cells in skeletal muscle. The quantitative data obtained in the present study indicate the presence of the protease in the cytosol fraction of all rat tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification, composition, and some properties of rat liver carbamyl phosphate synthetase (ammonia).

Hepatic microsomes prepared from vitamin E deficient and supplemented rats were analyzed for cytochrome P-450 content and drug metabolizing activity. Reduced levels of benzo[α]pyrene hydroxylase and ethylmorphine N-demethylase activities were observed in microsomes derived from rats fed a diet deficient in vitamin E compared to those of control rats. NADPH-mediated destruction of P-450, and pentobarbital and zoxazolamine sleeping times were similar in the two groups. Induction with 3-methylcholanthrene raised the levels of benzo[α]pyrene hydroxylase activity of both supplemented and deficient rats to the same absolute levels. No differences were noted in cytochrome P-450 or P-448 content between control and tocopherol deficient rats, nor did the activity of liver catalase differ between the two dietary groups. Thus, these studies did not demonstrate any impairment of heme protein synthesis in vitamin E deficient rats.     

Purification and some kinetic properties of rat liver glucosamine synthetase

1. Glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was purified about 300-fold from rat liver by two techniques. One procedure utilized the protective action of fructose 6-phosphate and gave a relatively stable preparation, the other yielded an unstable enzyme (half-life of about 20h), free of contaminant activities, on which kinetic experiments were performed. Although the properties of the two preparations showed slight differences, the unstabilized form could be converted into the stabilized form. 2. During preparation the enzyme retained its sensitivity to the feedback inhibitor, UDP-N-acetylglucosamine. 3. The reversibility of the enzyme-catalysed reaction could not be demonstrated. There was no apparent requirement for a cofactor. 4. The pH optimum was at 7.5, at which pH the reaction obeyed a Ping Pong Bi Bi rate equation. At pH values outside the range 6.9-7.6 and at temperatures below 29 degrees C the velocity was described by an ordered Bi Bi rate equation. 5. The molecular weight of the enzyme, determined by two procedures, was 360000-400000. 6. The aminotransferase was unable to utilize ammonia as a substrate.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

A kinetic analysis of biosynthesis and localization of a lysosome-associated membrane glycoprotein

The biosynthesis and subcellular distribution of a major lysosomal membrane glycoprotein of mouse embryo 3T3 cells, LAMP-1, have been examined by [35S]methionine pulse-labeling, sucrose density gradient fractionation, and oligosaccharide analysis. Mature LAMP-1, immunoprecipitated after labeling for 4 h, had a molecular mass of about 110,000 Da. It comigrated during sucrose density fractionation with lysosomal markers, consistent with previous electron microscopic evidence for its localization in lysosomal membranes. Precursor molecules, pulse-labeled for 5 min and extracted during the first 15 min of post-translational processing, were concentrated in the rough endoplasmic reticulum fraction as a species of 92,000 Da. Within 30 min after synthesis, LAMP-1 was found in fractions enriched in Golgi and lysosomal marker enzyme activities as the mature 110,000-Da glycoprotein. Oligosaccharide processing was complete by 1 h after synthesis, and the mature glycoprotein remained in a fraction bearing lysosomal markers. Treatment of the 92,000-Da precursor with endo-beta-N-acetyl-glucosaminidase H produced a core polypeptide of 43,000 Da. Pulse-labeling in the presence of tunicamycin yielded a 42,000-Da form of LAMP-1, which was converted within 30 min to a 43,000-Da molecule. Bio-Gel column chromatography and hexosamine/hexosaminitol analyses indicated that the mature 110,000-Da molecule contained both complex-type and high-mannose N-linked oligosaccharides.     

Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.     

Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase).

Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins

As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.     

Purification, biosynthesis and cellular localization of a major 125-kDa glycophosphatidylinositol-anchored membrane glycoprotein of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been shown to contain a major 125-kDa membrane glycoprotein which is anchored in the lipid bilayer by a glycophosphatidylinositol anchor. This protein was purified to near homogeneity and was used to raise a rabbit antibody. Biosynthesis of the 125-kDa protein was studied by immunoprecipitation of 35SO4-labeled material from wild-type cells or a secretion mutant (sec18) in which the vesicular traffic from the endoplasmic reticulum (ER) to the Golgi is blocked. The 125-kDa protein is first made in the ER as a 105-kDa precursor which already contains a glycophosphatidylinositol anchor and which is slowly transformed into the 125-kDa form upon chase (t1/2 approximately 10-15 min). The 105-kDa precursor can be reduced to an 83-kDa form by the enzymatic removal of N-glycans. The removal of N-glycans from the mature 125-kDa protein yields a 95-kDa species. Thus, removal of the N-glycans does not reduce the ER and mature forms to the same molecular mass, indicating that not only elongation of N-glycans but also another post-translational modification takes place during maturation. Selective tagging of surface proteins by treatment of 35SO4-labeled cells with trinitrobenzene sulfonic acid at 0 C followed by immunoprecipitation of the tagged proteins shows that the 125-kDa protein, but not the 105-kDa precursor, becomes transported to the cell surface. This tagging of cells after various lengths of chase also shows that the surface appearance of the protein is biphasic with about one half of the mature 125-kDa protein remaining intracellular for over 2 h. Glycosylation and/or glycophosphatidylinositol anchor addition is important for the stability of the 125-kDa protein since the protein remains undetectable in sec53, a temperature-sensitive mutant which does not make GDP-mannose at 37 C and does not add glycophosphatidylinositol anchors at 37 degrees C.