Development and Regional Distribution of Methionine Synthetase in Rabbit Brain

The development and regional distribution of methionine synthetase (EC 2.1.1.13) in rabbit brain was determined. In adult rabbits, the specific activity (units per milligram protein) of methionine synthetase in cortex, cerebellum, brain stem, and corpus striatum was comparable to the specific activity in whole brain (0.5 units/mg). In the first few weeks of life, the specific activity of methionine synthetase in whole rabbit brain declined from a value of 1.1 units/mg at 1 day of age to 0.5 units/mg at 6–10 weeks. Two-year-old rabbits had 0.6 units/mg in whole brain. These results show that: (a) methionine synthetase is distributed widely in mammalian brain and (b) methionine synthetase activity in brain declines relatively little with development.     

Acyl-coenzyme-A synthetase I from Candida lipolytica. Purification, properties and immunochemical studies.

Acyl-coenzyme-A synthetase I from Candida lipolytica has been purified to homogeneity as evidenced by polyacrylamide gel electrophoresis in the presence and absence of dodecylsulfate as well as by Ouchterlony double-diffusion analysis. The purification procedure involves resolution of cellular particles with Triton X-100 and chromatography on phosphocellulose, Blue-Sepharose and Sephadex G-100. The purified enzyme exhibits a specific activity of 20--24 U/mg protein at 25 degree C, which is about 100-fold higher than those of long-chain acyl-CoA synthetases hitherto reported. The molecular weight of the enzyme has been estimated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate to be approximately 84 000. The enzyme is specific for fatty acids with 14--18 carbon atoms regardless of the degree of unsaturation. Studies with the use of specific antibody to acyl-CoA synthetase I have indicated that this enzyme is immunochemically distinguishable from acyl-CoA synthetase II.     

Comparison of free and ribosome-bound phenylalanyl-tRNA synthetase from rabbit reticulocytes.

Phenylalanyl-tRNA synthetase (l-phenylalanine:tRNA ligase [AMP], EC 6.1.1.b) from the ribosomal and the postribosomal cell supernatant fractions of rabbit reticulocytes were purified separately and characterized. Phenylalanyl-tRNA synthetase from the ribosomal fraction was purified 114-fold to a final specific activity of 1603 units/mg and is approximately 90% pure. Phenylalanyl-tRNA synthetase from the postribosomal supernatant fraction was purified 4186-fold to a final specific activity of 247 units/mg. The enzymes from the two fractions appear to be identical based on their elution from various chromatographic media, sedimentation coefficient, pH, Mg²⁺, and K⁺ optima, and heat stability. Phenylalanyl-tRNA synthetase from rabbit reticulocytes has a molecular weight of approximately 245,000 with an α₂β₂ subunit structure. The molecular weights of the subunits are 57,000 and 67,200.     

Purification of bovine heart glycogen synthase

Glycogen synthase was purified to apparent homogeneity from bovine heart muscle by a procedure involving precipitation of the enzyme in the presence of added glycogen by polyethylene glycol, chromatography on DEAE-Sephacel, and high-speed centrifugation through a sucrose-containing buffer. The enzyme was maintained in the presence of glycogen during the isolation procedure. Glycogen synthase I and D preparations were obtained having specific activities of 21-25 and 30-35 units/mg protein at pH 7.8 and 30 degrees C and having activity ratios of 0.5-0.6 and 0.05-0.10, respectively, when assayed in the absence and in the presence of glucose 6-P.     

Mechanisms of action of NIP71 on N-myristoyltransferase activity

N-Myristoyl-CoA:protein N-myristoyltransferase (NMT) is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates. NMT was highly purified from bovine brain by procedures involving sequential column chromatography on DEAE-Sepharose CL-6B, phosphocellulose, hydroxylapatite, and mono S and mono Q f.p.l.c.. The highly purified NMT (termed NMT·II) possessed high specific activity with peptide substrates derived from the N-terminal sequences of the cAMP-dependent protein kinase and pp60^src (29,800 and 47,600 pmol N-myristoylpeptide formed/min/mg, respectively), intermediate activity with a peptide based on the N-terminal sequence of a viral structural protein (l) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence ofmyristoylatedalanine-richC-kinasesubstrate (MARCKS; 1500 pmol myristoylpeptide formed/min/mg). An NMT protein inhibitor (NIP₇₁) isolated from the particulate fraction of bovine brain (King MJ and Sharma RK: Biochem J 291635-639, 1993) potently inhibited highly purified NMT activity (IC₅₀ 23.7 nM). A minor NMT activity (NMT·PU; 30% total NMT activity), which failed to bind to phosphocellulose, was insensitive to NIP₇₁ inhibition. Inhibition of NMT was observed to be via mixed inhibition with respect to both the myristoyl-CoA and peptide substrates with NIP₇₁ having an apparent higher affinity for NMT than the NMT·myristoyl·CoA complex. Inhibition by NIP₇₁ at subsaturating concentrations of myristolyl-CoA and peptide resulted in a sigmoidal pattern of inhibition indicating that bovine brain possesses a potent and delicate on/off switch to control NMT activity.Abbreviations NMT N-myristoyl-CoA:protein N-myristoyltransferase - NMT·I mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak I - NMT·II mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak II - NMT·III mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak III - NIP₇₁ 71 kDa heat-stable N-myristoyltransferase inhibitor protein     

Purification and properties of prostaglandin D synthetase from rat brain.

The prostaglandin D synthetase system was isolated from rat brain. Prostaglandin endoperoxide synthetase solubilized from a microsomal fraction catalyzed the conversion of arachidonic acid to prostaglandin H2 in the presence of heme and tryptophan. Prostaglandin D synthetase (prostaglandin endoperoxidase-D isomerase) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2 was found predominantly in a cytosol fraction and was purified to apparent homogeneity with a specific activity of 1.7 mumol/min/mg of protein at 24 degrees C. The enzyme also acted upon prostaglandin G2 and produced a compound presumed to be 15-hydroperoxy-prostaglandin D2. Glutathione was not required for the enzyme reaction, but the enzyme was stabilized by thiol compounds including glutathione. The enzyme was inhibited by p-chloromercuribenzoic acid in a reversible manner. The purified enzyme was essentially free of the glutathione S-transferase activity which was found in the cytosol of brain.     

Purification and characterization of human factor II

Human plasma Factor II has been purified approximately 800-fold by a combination of barium citrate adsorption, ion-exchange chromatography and preparative polyacrylamide gel electrophoresis. The procedure is relatively simple and results in excellent yields of purified Factor II essentially free of Factor X activity. The purified factor behaved as a single component by analytical polyacrylamide gel disc electrophoresis at pH 8.9. No Factor V, VII or IX activity was detected in the purified Factor II. Its molecular weight was 7200±3000 as determined by analytical ultracentrifugation, electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on Bio-Gel P-200. An apparent molecular weight of 90 000–100 000 was observed on calibrated columns of Sephadex G-100, G-150, and G-200. The specific activity of human factor II was approximately 1300 N.I.H. units/mg as determined by the two-stage assay and 7 Ortho units/mg by the one stage assay. The purified protein contained by weight 2.8% neutral hexose, 2.3% sialic acids and 3.1% hexosamines.     

Purification of isopenicillin N synthetase from Streptomyces clavuligerus

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 X 10(-3) units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.     

Trial of purifying tumor degenerating factor of human fibroblasts

Tumor-degenerating factor (TDF) with the specific activity of 2.9 units/mg of protein was produced and purified by several chromatographies. The specific activity was increased to 302 units/mg of protein by DEAE-Sephadex A-50 chromatography, repeated twice. Then, this preparation was purified to the specific activity of 2,040 units/mg of protein with recovery rate of 16.6% by Con A-Sepharose and CM-Sephadex C-50 chromatographies. Finally, the specific activity was increased to 9,010 units/mg of protein with the final recovery rate of 14.6% by Blue Sepharose CL-6B chromatography.     

Regulation of glycogen synthetase. Specificity and stoichiometry of phosphorylation of the skeletal muscle enzyme by cyclic 3':5'-AMP-dependent protein kinase.

Complete conversion of skeletal muscle glycogen synthetase from the I form to the D form requires incorporation of 2 mol of phosphate per enzyme subunit (90,000 g). Incubation of sythetase I with low concentrations of adenosine 3':5'-monophosphate(cAMP)-dependent protein kinase (10 units/ml) and ATP (0.1 to 0.3 mM) plus magnesium acetate (10 mM) results in incorporation within 1/2 hour of 1 mol of phosphate persubunit concomitant with a decrease in the synthetase activity ratio (minus glucose-6-P/plus glucose-6-P) from 0.85 to 0.25. Further incubation for 6 hours does not greatly increase the phosphate content of the synthetase or promote conversion to the D form. This level of phosphorylation is not increased by raising the concentration of protein kinase to 150 units/ml and is not influenced by the presence of glucose-6-P, UDP-glucose, or glycogen. However, at protein kinase concentrations of 10,000 to 30,000 units/ml a second mol of phosphate is incorporated per subunit, and the sythetase activity ratio decreases to 0.05 or less. In addition to the 2 mol of phosphate persubunit which are required for formation of sythetase D, further phosphorylation can be observed which is not associated with changes in synthetase activity. This phosphorylation occurs at a slow rate, is increased by raising the ATP concentration to 2 to 4mM, and is not blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. These data indicate that skeletal muscle glycogen synthetase contains multiple phosphorylation sites only two of which are involved in the synthetase I to D conversion.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

Ferredoxin-dependent Nitrite Reductase from Spinach Leaves

A ferredoxin-dependent nitrite reductase from Spinacea oleracea was purified approximately 180-fold, with a specific activity of 285 units/mg protein. This purified enzyme also had methyl viologen-dependent nitrite reductase activity, with a specific activity of 164 units/mg protein. After disc electrophoresis with polyacrylamide gel, the purified enzyme showed one major and one minor protein band.

The molecular weight of the enzyme was estimated to be 86,000 from Ultrogel filtration. This purified enzyme in oxidized form had absorption peaks at 278, 390, 573 and 690 nm. The absorbance ratios, A₃₉₀: A₂₇₈ and A₆₇₃: A₃₉₀ were 0.61 and 0.37, respectively.

By applying the purified enzyme to DEAE-Sephadex A–50 column chromatography, the ferredoxin-dependent nitrite reductase activity was selectively decreased. However, the methyl viologen-dependent nitrite reductase activity was increased, with a specific activity of 391 units/mg protein. This modified enzyme was homogeneous by disc electrophoresis with polyacrylamide gel.     

Purification and characterization of two novel arginine aminopeptidases from Streptococcus mitis ATCC 9811

Two novel aminopeptidases (I and II) which have specificity for amino-terminal arginine residues and strong sensitivity to divalent cations were purified from Streptococcus mitis ATCC 9811 by a procedure that involved treatment with a lytic enzyme for bacterial cell walls, followed by a series of chromatographies. Enzyme I was obtained as a homogeneous protein as judged by polyacrylamide gel electrophoresis and had a specific activity of 484.8 units per mg protein using L-arginine-2-naphthylamide as substrate; its Km value was 2.6 X 10(-5) M. The molecular weight was estimated to be 62,000, and its isoelectric point was pH 4.4. Enzyme II was purified to a specific activity of 128.0 units per mg protein and had a Km value of 3.8 X 10(-5) M. The molecular weight was estimated to be 360,000, and its isoelectric point was pH 5.7. The pH optima of enzymes I and II were 8.6 and 7.6, respectively. Both enzymes were inactivated by sulfhydryl reagents and metal ions but were markedly activated by EDTA. The chloride ion had an inhibitory rather than a stimulatory effect on the activity of both enzymes. Substrate specificity studies indicated that both the enzymes specifically hydrolyze N-terminal arginine residues from a-aminoacyl 2-naphthylamides and peptides, but they could not attack the L-arginyl-L-prolyl-peptide.     

Purification of α-galactosidase from coconut

α-Galactosidase from coconut endosperm was purified to homogeneity with a 490-fold increase in specific activity. The yield was 70%, and the specific activity was 24.5 units/mg protein. The purification procedure included extraction, acidification, ammonium sulphate fractionation and hydrophobic chromatography. The hydrophobic gel (Sepharose-4B-capranilide) had a capacity of 0.63 mg of α-galactosidase per ml of gel. Purified α-galactosidase was a glycoprotein with a carbohydrate content of 12%. The molar extinction coefficient was 8.7 x 10⁴/M/cm.     

Purification and properties of trehalase from monkey small intestine

Brush border membrane trehalase was purified from monkey small intestine by a procedure which includes solubilisation by Triton X-100, ammonium sulphate fractionation, and chromatography on DE-52 and hydroxyapatite. The purified enzyme had a specific activity of 11 units/mg protein and was purified 140-fold. The enzyme showed a single protein band on Polyacrylamide gel electrophoresis. It had aK ^m value of 17.4 mM for trehalose and a V_max of 1.33 units. Sucrose and Tris acted as competitive inhibitors of the enzyme.     

9 alpha,11 beta-prostaglandin F2 formation in various bovine tissues. Different isozymes of prostaglandin D2 11-ketoreductase, contribution of prostaglandin F synthetase and its cellular localization

9 alpha,11 beta-prostaglandin F2 was formed from prostaglandin D2 by its 11-ketoreductases in 100,000 x g supernatants of various bovine tissues in the presence of an NADPH-generating system. The reductase activities were high in liver (51.09 nmol/h/mg of protein), lung (24.99), and spleen (14.20); moderate in heart and pancreas (3.09-3.61); weak in stomach, intestine, colon, kidney, uterus, adrenal gland, and thymus (0.11-2.63); and undetectable in brain, retina, carotid artery, and blood (less than 0.10). No formation of prostaglandin F2 alpha from prostaglandin D2 was detected in all tissues. In immunotitration analyses with a polyclonal antibody specific for prostaglandin F synthetase, the reductase activities in lung and spleen showed identical titration curves to that of the purified synthetase and decreased to less than 15% of the initial activity under the condition of antibody excess. Prostaglandin F synthetase-immunoreactive protein in these two tissues showed peptide fingerprints identical to that of the purified enzyme after partial digestion with Staphylococcus aureus V8 protease. The antibody was partially cross-reactive to the reductase in liver (about 20% of that to the synthetase) but not to the reductase(s) in other tissues. The Km value for prostaglandin D2 of the reductase activity was the same in lung and spleen as that of the purified prostaglandin F synthetase (120 microM) but differed in liver (6 microM), heart, and pancreas (15 microM). The predominant distribution of prostaglandin F synthetase in lung and spleen was confirmed by radioimmunoassay (2.8 and 1.0 micrograms/mg protein, respectively) and Northern blot analyses. In immunoperoxidase staining, this enzyme was localized in alveolar interstitial cells and nonciliated epithelial cells in lung, histiocytes and/or dendritic cells in spleen, and a few interstitial cells in kidney and adrenal cortex.     

Rapid Purification of Enzymes of Fatty Acid Biosynthesis from Rat Adipose Tissue

Abstract

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protien kinases.     

Purification and properties of barley leaf ribonuclease

The principal ribonuclease from young barley plants was purified 29 200-fold by a six-step procedure. The enzyme showed a high specific activity (15 5OO ΔA₂₆₀ units/min/mg protein) and a molecular weight of about 25 000 was indicated by gel filtration and equilibrium sedimentation. Kinetic analysis of the cleavage of dinucleoside monophosphates and of yeast RNA indicated a base preference of Gua > Ade ≥ Ura ? Cyt, and was sensitive to the base located on either side of the phosphodiester bond. The enzyme resembles the Type I class of plant ribonucleases (E.C. 2.7.7.x).     

Purification of human gamma-interferon to essential homogeneity and its biochemical characterization

A multistep procedure has been developed which enables human gamma-interferon (HuIFN-gamma) to be purified to essential homogeneity. The procedure takes advantage of a modification of a previously described sequential chromatographic technique [Braude, I.A. (1983) Prep. Biochem. 13, 177-190] and the high isoelectric point of HuIFN-gamma (pH 9.5-9.8). The steps include Controlled Pore Glass adsorption chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, cation-exchange chromatography, and gel filtration chromatography. The purified HuIFN-gamma had a specific activity of 5.9 X 10(7) units/mg. This represents a purification of more than 70 000-fold and a 33% recovery. In addition, one gel filtration fraction had a specific activity of 2.5 X 10(8) units/mg. This represents a purification of greater than 300 000-fold and a recovery of greater than 17%. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown to be composed of one major 26-kilodalton (kDa) species and four minor species of 74, 67, 56, and 22 kDa. Analysis of this material with anti-HuIFN-gamma monoclonal antibody immunoabsorbent columns indicates that both the 26- and the 22-kDa species are HuIFN-gamma. Thus, the final product is essentially homogeneous (90-92% HuIFN-gamma), and the specific activity of pure HuIFN-gamma is approximately (2.7-2.8) X 10(8) units/mg of protein. Finally, the 26- and 22-kDa moieties are shown to be similar, if not identical, proteins as judged by amino acid and sequence analyses.     

Purification and properties of pyruvate kinase from liver of the flounder (Platichthys flesus L.)

Two forms of pyruvate kinase, PK I and PK II, have been demonstrated in flounder liver. PK I, purified 991-fold to a specific activity of 105 units per mg of protein, has an unusually high molecular weight of about 2 X 10(6). PK II, purified 172-fold to a specific activity of 16.5 units per mg of protein, has a molecular weight of 210,000 when determined on a sucrose gradient but of 300,000 when derived from gel chromatography. PK I and PK II differ in sensitivity to the inhibitor L-phenylalanine, a fact which is used to evaluate the amount of each of them in a mixture. pH optimum for both forms is 6-6.6. PK I and PK II behave different in an Arrhenius plot--PK II showing a transition at 21 degrees C.