Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme)

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.     

Guanosine triphosphate: 5-hydroxylysine phosphotransferase in rat kidney cortex.

An enzyme which catalyzes the transfer of the gamma-phosphate from GTP onto 5-hydroxylysine was partially purified from rat kidney cortex by means of acid precipitation and DEAE-Sephadex A-50 column chromatography. The enzyme activity was assayed by measuring the transfer of [32P] from gamma-[32P]-GTP to materials not adsorbed by charcoal. This partially purified enzyme showed essentially no GTP phosphohydrolase activity and an optimal pH of 8.0. An apparent Km of about 23.8 mumol/1 was obtained with respect to 5-hydroxylysine. Mg2+ was required for the activity of this enzyme. Ethanolamine, L-lysine, L-ornithine and choline inhibited the enzyme but L-threonine, L-serine and hydroxy-L-proline did not. None of these compounds severed as substrate for this enzyme.     

Some characteristics of sn-glycero-3-phosphocholine diesterases from rat brain.

1. sn-Glycero-3-phosphocholine diesterase activities, glycerophosphohydrolase (EC 3.1.4.2) and choline phosphohydrolase (EC 3.1.4.38) from rat brain have been partially purified and characterized using sn-glycere-3-[32P]phosphocholine as substrate and separating the reaction products by anion-exchange chromatography and ionophoresis. 2. Rat brain contained particulate (75%) and soluble (25%) activity from both diesterases. No difference in pH optimum or metal ion requirement for the particulate compared to the soluble enzymes was observed. 3. Glycerophosphohydrolase (EC 3.1.4.2) was purified 60-fold, choline phosphohydrolase (EC E.1.4.38) 120-fold from rat brain supernatant fraction by DEAE-cellulose ion-exchange chromatography and sucrose density gradient centrifugation. The density gradient results in conjunction with dodecyl sulphate-polyacrylamide gel disc electrophoresis yielded molecular weight estimates of 230 000 (monomer 62 000) for choline phosphohydrolase and 120 000 (monomer 70 000) for glycerophosphohydrolase (EC 3.1.4.2). 4. Glycerophosphohydrolase (EC 3.1.4.2) has a pH optimum of 8.9 and a Km for sn-glycero-3-phosphocholine of 0.6 mM. The enzyme is inhibited by EDTA and reactivated by Ca2+. Choline phosphohydrolase (EC 3.1.4.38) has pH optimum 10.5, a Km of 2 mM and is unaffected by EDTA. Both enzymes require Ca2+ for maximum activity.     

Purification and properties of a mannosyltransferase solubilized from mitochondrial outer membranes

The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated choline-containing lipids such as phosphatidylcholine, lysophatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl posphate-mannose. The partially purified mannosyltrasnferase exhibits a K_m of 1.33 μM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient certrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.     

Partial purification and some properties of gamma-glutamyl transpeptidase from human bile.

1. Gamma-Glutamyl transpepetidase ((5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2) from human bile has been partially purified using protamine sulphate treatment, DEAE-cellulose chromatography and Sephadex G-200 filtration. The procedure resulted in 150-fold increase in specific acitivity with a 37% yield. 2. The partially purified enzyme showed a single zone of enzyme activity by polyacrylamide gel electrophoresis and eluted in the inner volume of Sephadex G-200. 3. The enzyme had a pH optimum of 8.1 and Km of 1.52 mM using gamma-glutamyl p-nitroanilide as substrate. 4. The effects of cations and different gamma-glutamyl acceptors on the activity of the enzyme are reported. 5. As bile gamma-glutamyl transpeptidase appears to be soluble in the absence of detergents, it is suggested that bile may prove to be a useful source for further studies of the kinetic properties and physiological role of human gamma-glutamyl transpeptidase.     

On the nature of the interaction between 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid and microsomal glucose-6-phosphatase. Evidence for the involvement of sulfhydryl groups of the phosphohydrolase

The effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (DIDS) on microsomal glucose 6-phosphate hydrolysis has been reinvestigated and characterized in order to elucidate the topological and functional properties of the interacting sites of the glucose-6-phosphatase. The studies were performed on microsomal membranes, partially purified and reconstituted glucose-6-phosphatase preparations and show the following. (a) DIDS inhibits activity of the glucose-6-phosphatase of native microsomes as well as the partially purified glucose-6-phosphatase. (b) Inhibition is reversed when the microsomes and the partially purified phosphohydrolase, incorporated into asolectin liposomes, are modified with Triton X-114. (c) Treatment of native microsomes with DIDS and the following purification of glucose-6-phosphatase from these labeled membranes leads to an enzyme preparation which is labeled and inhibited by DIDS. (d) Preincubation of native microsomes or partially purified glucose-6-phosphatase with a 3000-fold excess of glucose 6-phosphate cannot prevent the DIDS-induced inhibition. (e) Inhibition of glucose-6-phosphatase by DIDS is completely prevented when reactive sulfhydryl groups of the phosphohydrolase are blocked by p-mecuribenzoate. (f) Reactivation of enzyme activity is obtained when DIDS-labeled microsomes are incubated with 2-mercaptoethanol or dithiothreitol. Therefore, we conclude that inhibition of microsomal glucose 6-phosphate hydrolysis by DIDS cannot result from binding of this agent to a putative glucose-6-phosphate-carrier protein. Our results rather suggest that inhibition is caused by chemical modification of sulfhydryl groups of the integral phosphohydrolase accessible to DIDS attack itself. An easy interpretation of these results can be obtained on the basis of a modified conformational model representing the glucose-6-phosphatase as an integral channel-protein located within the hydrophobic interior of the microsomal membrane [Schulze et al. (1986) J. Biol. Chem. 261, 16,571-16,578].     

Human alpha-fucosidase. Purification and properties.

Human placental alpha-fucosidase (EC 3.2.1.51) has been extensively purified and partially characterized with respect to kinetic and structured properties. Although the enzyme seems to be separated by DEAE-cellulose chromatography in two forms which differ in their molecular weight and thermostability, an interconversion between the two forms takes place during storage and/or electrofocusing so that the same peaks of activity, revealed by the latter technique, are found before and after DEAE-cellulose chrome. The heterogeneous peaks of activity revealed by isoelectrofocusing show a reproducible pattern in the different tissues examined, except in serum where their pI values are consistently more acidic.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

Partial characterization of a soluble ATPase from pea cotyledon mitochondria.

A partially purified soluble ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pea cotyledon mitochondria was characterized. Inhibition patterns with azide, NaF, and cold, and a stimulation by 2,4-dinitrophenol were typical of F1-ATPases from mammalian mitochondria. The enzyme hydrolysed GTP, ITP, and ATP, but not CTP, UTP, ADP, or IDP. ATPase and ITPase activities were strongly inhibited by ADP and to a lesser extent by IDP. Distinctive properties of the pea mitochondrial enzyme were activation by high concentrations of CaCl2 and stimulation by NaCl.     

Rat testis mitochondrial adenylate cyclase. Partial purification and characterization.

1. Adenylate cyclase (EC 4.6.1.1) from rat testis mitochondria has been solubilized by treatment with the non-ionic detergent Lubrol PX. The soluble enzyme was further purified by DEAE-cellulose chromatography. 2. The specific activity of the adenylate cyclase eluted from the DEAE-cellulose column was found to be four times higher than that of an intact mitochondrial preparation. At this step the enzyme shows a sedimentation coefficient of 4.2 S and a diffusion coefficient (D) of 3.12 - 10- minus 7 cm-2/sec. 3. Solubilization of the adenylate cyclase resulted in loss of responsiveness to gonadotrophic hormones. Addition of phosphatidylserine to the soluble preparation partially restored the activation of adenylate cyclase by human chorionic gonadotrophin. 4. The results of this study suggest that the activity of the adenylate cyclase may be dependent on the membrane-bound phospholipids and that the enzyme attached to the mitochondrial membranes has some properties which are similar to the adenylate cyclase found to be associated with other membrane systems of the cell.?     

Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver.

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.     

Sterolsulphate sulphohydrolase from human placenta microsomes--30 kDa molecular weight form of cholesterol sulphate sulphohydrolase

The cholesterol sulphate sulphohydrolase (CHS-ase) exhibiting molecular weight of 30 kDa was purified from human placenta microsomes. The microsomal proteins were extracted with 0.5% Triton X-100. The DEAE-cellulose chromatography of the solubilized microsomal proteins, performed at pH 7.6 allowed to separate two enzymatically active fractions. One of them was associated with the protein fraction unbound by DEAE-cellulose, the other was tightly bound by ion exchanger. The 30 kDa cholesterol sulphate sulphohydrolase was purified to homogenity from the protein fraction tightly bound by DEAE-cellulose. The highly purified enzyme preparation (specific activity 385 nmol min(-1)mg(-1) of protein) exhibited optimal activity at pH 6.4, the K(m) was established to be 6.7 x 10(-6)M, the pI value was 7.4. The 30 kDa cholesterol sulphate sulphohydrolase, in contrast to the CHS-ase form originated from the protein fraction unbound by DEAE-cellulose, was not sensitive to alkaline phosphatase treatment and phosphohydrolase inhibitors. The effects of steroids, -SH reacting agents and sulphohydrolase inhibitors on the enzyme activity were tested.     

A member of a new class of GTP cyclohydrolases produces formylaminopyrimidine nucleotide monophosphates

The hyperthermophilic euryarchaeon Methanococcus jannaschii has no recognizable homologues of the canonical GTP cyclohydrolase enzymes that are required for riboflavin and pteridine biosyntheses. Instead, it uses a new type of thermostable GTP cyclohydrolase enzyme that produces 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone ribonucleotide monophosphate and inorganic phosphate. Whereas canonical GTP cyclohydrolases produce this formylamino-pyrimidine nucleotide as a reaction intermediate, this compound is shown to be an end product of the purified recombinant M.jannaschii enzyme. Unlike other enzymes that hydrolyze the alpha-beta phosphate anhydride bond of GTP, this new enzyme completely hydrolyzes pyrophosphate to inorganic phosphate. As a result, the enzyme has a steady-state turnover of 21 min(-)(1), which is much faster than those of canonical GTP cyclohydrolase enzymes. The effects of substrate analogues and inhibitors suggest that the GTP cyclohydrolase and pyrophosphate phosphohydrolase activities occur at independent sites, although both activities depend on Mg(2+).     

Ribosome-associated cyclic nucleotide-independent protein kinase of Artemia salina cryptobiotic gastrulae

An extra-ribosomal cAMP-independent protein kinase from cryptobiotic embryos of Artemia salina has been purified to near homogeneity by gel filtration on Bio-Gel A-0.5 m, ion-exchange chromatography on DEAE-cellulose and phosphocellulose P11 and affinity chromatography on casein-Sepharose 4B and ATP-agarose. The enzymatic activity has a broad optimum at pH 7-8. Maximal activity is obtained in the presence of 5-6 mM MgCl2. The activity is inhibited by Mn2+, Ca2+ and K+. The enzyme has an Mr of 127 000, utilizes both ATP and GTP as phosphoryl donors and is completely inhibited by heparin and poly(L-glutamic acid). According to its properties, the enzyme can be classified as a casein kinase type II. Although the enzyme is associated with ribosomes, ribosomal proteins are not among the main substrates. The kinase is able to phosphorylate both the alpha and the beta subunits of initiation factor eIF2 using ATP or GTP as phosphoryl donors. The function of phosphorylation in the initiation of protein synthesis is discussed.     

Polyadenylate polymerase from cytoplasm and nuclei of N.I.H.-Swiss mouse embryos.

Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.     

Partial purification of 2,3-oxidosqualene-lanosterol cyclase from hog-liver. Evidence for a functional thiol residue

2,3-Oxidosqualene-lanosterol cyclase is an intrinsic microsomal protein which can be solubilized by ionic (deoxycholate) and nonionic (emulphogene) detergents with good yields. The hog-liver microsomal cyclase was purified approximately 140-fold by chromatography on DEAE-cellulose and hydroxylapatite. The partially purified enzyme was inactivated by N-ethylmaleimide, following pseudo-first order kinetics, indicating that a cysteine residue is essential for activity.     

Diadenosine tetraphosphatase from human leukemia cells. Purification to homogeneity and partial characterization

Diadenosine tetraphosphatase, an enzyme splitting diadenosine tetraphosphate to AMP and ATP, has been purified to apparent homogeneity from a permanent cell line derived from a leukemic child. The purification procedure consisted of fractionation by ammonium sulfate precipitation, followed by Sephacryl 200 and DEAE-cellulose chromatography, and finally a differential membrane filtration. The enzyme is a single polypeptide chain of Mr = 17,500 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent molecular weight of the native enzyme was calculated as 20,000 from gel filtration data. The apparent Km for Ap4A was 0.5 microM as determined by two independent kinetic assays. None of the following compounds were substrates of the enzyme: diadenosine triphosphate, NAD, nucleoside 5'-phosphates (AMP, ATP, GDP, GTP, and UTP). The enzyme had optimal activity in the presence of 1 mM Mg2+, showing no activity in the presence of EDTA.     

Identification and characterization of a DNase induced by Epstein-Barr virus.

The diterpene ester promoter of mouse skin tumors, 12-O-tetradecanoyl-phorbol-13-acetate, induced a DNase activity in the Epstein-Barr virus-producer cell line P3HR-1. The elution patterns of the enzyme from DEAE-cellulose, phosphocellulose, and DNA-cellulose columns were different from virus-associated DNA polymerase activity. The partially purified activity could be neutralized to the extent of 90% by sera of patients with nasopharyngeal carcinoma. Purified immunoglobulin G from sera of nasopharyngeal carcinoma patients inhibited this enzyme and that obtained from superinfected Raji cells to the same extent. The partially purified enzyme preferred native DNA as a substrate over denatured DNA and 3'-terminally labeled activated calf thymus DNA. The activity was inhibited by high ionic strength. Phosphonoformic acid did not have any effect on this enzyme activity.     

Purification and properties of glutamate dehydrogenase from the mantle muscle of the squid, Loligo pealeii. Role of the enzyme in energy production from amino acids.

1. The activity of glutamate dehydrogenase was measured in the tissues of the squid, Loligo pealeii. The enzyme occurs in high activity in digestive pouch, systemic heart, and all muscle tissues. 2. Glutamate dehydrogenase from mantle muscle is located intra-mitochondrially, has a molecular weight of 310,000, and is electrophoretically similar to the enzyme from all other squid tissues. 3. The enzyme from mantle muscle was purified 40-fold by elution from DEAE-cellulose and used for kinetic studies. The enzyme is NAD+-specific, activated by ADP, AMP, and leucine, and inhibited by GTP, GDP, ATP, and reaction products (in particular NADH). 4. Squid glutamate dehydrogenase shows an almost absolute dependence on ADP. The purified enzyme is activated over 100-fold by saturating concentrations of ADP (Ka = 0,75 7M); The pH optima are also altered significantly by ADP. 5. The enzyme appears to be kinetically adapted to favour glutamate oxidation in comparison to glutamate dehydrogenase from other resources. The evidence indicates that the primary role of glutamate dehydrogenase in squid mantle muscle is in regulating the catabolism of amino acids for energy production.