Characterization and partial purification of a novel mannosylphosphodolichol phosphodiesterase from chicken liver microsomes

Mannosylphosphodolichol phosphodiesterase, which catalyzes the release of mannose from mannosylphosphodolichol, was solubilized from chicken liver microsomes by treatment with the non-ionic detergent, Emulgen 909. The enzyme was partially purified using ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration on Sepharose 6B. The enzyme showed absolute requirement for sulfhydryl reducing agents. The enzyme activity was stimulated by the addition of CaCl2 and Emulgen 909 and exhibited a pH optimum around 5.3. The Km value for mannosylphosphodolichol was found to be 0.43 microM. The activity was competitively inhibited by dolichyl phosphate and dolichol and the Ki value for dolichyl phosphate was estimated to be 12.5 microM. The purified preparation had no activity toward N-acetylglucosaminyldiphosphodolichol, glucosylphosphodolichol, mannose 1-phosphate, or artificial substrates for mannosidases, glucosidases, acid phosphatase, and acid phosphodiesterase. A heat-stable factor which stabilizes the mannosylphosphodolichol phosphodiesterase was separated from the enzyme by DEAE-cellulose chromatography. It was precipitated by trichloroacetic acid and not extracted into lipid solvents. The separation resulted in the complete loss of the enzyme activity and the restoration of the activity was not observed when the factor was added back to the enzyme solution.     

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.     

GDPmannose dolicholphosphate mannosyltransferase of chicken liver mitochondria

Chicken liver mitochondria contain enzymes for the dolichol cycle. GDPmannose dolicholphosphate mannosyltransferase has been solubilized with Emulgen 909 and purified. The purified enzyme was not homogeneous, but highly specific for GDPmannose and dolichyl phosphate. The enzyme activity was stimulated by MgCl2 (3 mM optimum) and exhibited a pH optimum at around 7.2. Bisubstrate kinetic analysis indicated that the enzyme follows a sequential mechanism. The Km values for GDPmannose and dolichyl phosphate were 0.43 and 14.3 microM, respectively. The purified enzyme was labile and lost its activity on storage at 0 degree C overnight or incubation at 30 degrees C or higher temperature. Inactivation could be prevented by the addition of heat-denatured mitochondrial extract. Further investigation revealed that phospholipids and dolichyl phosphate are responsible for the stabilization. Single addition of either phospholipid or dolichyl phosphate showed little activity, but the combination of these lipids enhanced the stabilizing activity greatly. Eight naturally occurring phospholipids were tested and found to be effective in combination with dolichyl phosphate. Among these, sphingomyelin was the most effective. Dolichol could partially substitute dolichyl phosphate but worked at higher concentrations.     

Purification and properties of sterol: UDPG glucosyltransferase in cell culture of Digitalis purpurea

Sterol: UDPG glucosyltransferase was isolated for the first time from cell culture. Digitalis purpurea cultured cells had 2–5 times higher activity than that of the original plant. The enzyme in the particulate fraction was purified 70.2-fold from cell culture and 76-fold from the plant by cellular fractionation and column chromatography. The properties of purified enzyme from cultured cells were similar to those of the enzyme from the intact plant. The substrate specificity was the highest for a phytosterol.     

Preparation of antibodies to chick-embryo galactosylhydroxylysyl glucosyltransferase and their use for an immunological characterization of the enzyme of collagen synthesis

Antibodies were prepared against chick-embryo galactosylhydroxylysyl glucosyltransferase and further purified by immunoaffinity chromatography. The antibodies gave a single precipitation line of identity by double immunodiffusion against crude or pure chick-embryo glucosyltransferase. The ability of the antibody to precipitate the transferase was not altered by destroying the secondary structure of the enzyme. The antibody also inhibited the enzyme activity. The degree of inhibition was higher with denatured citrate-soluble rat skin collagen as the substrate than with gelatinized rat skin insoluble collagen or free galactosylhydroxylysine. The cross-reactivity of the glucosyltransferase between different species was low when studied by double immunodiffusion or inhibition kinetics. The antiserum showed no detectable cross-reactivity against other intracellular enzymes of collagen biosynthesis. A line of complete identity was found in double immunodiffusion between the transferases from whole chick embryos and chick embryo tendon, kidney and cartilage. Inhibition by the antiserum of the enzyme from chick embryo tissues synthesizing different collagen types was relatively similar. The data do not support the hypothesis that galactosylhydroxylysyl glucosyltransferase has isoenzymes with markedly different specific activities or immunological properties.     

Partial purification and properties of a murine plasmacytoma glucosyltransferase

The enzyme UDP-glucose:dolichylphosphate glucosyltransferase has been purified 1700-fold from MOPC-315 plasmacytoma tissue. The purification combines differential detergent extraction of purified rough endoplasmic reticulum with subsequent ion exchange chromatography, dye affinity chromatography, and hydroxylapatite chromatography of the extract. The partially purified glucosyltransferase exhibits a Km of 0.79 microM for UDP-Glc and a Km of 0.65 microM for dolicholphosphate in the presence of 4 mg/ml of phosphatidylcholine. The reaction is dependent upon the addition of exogenous dolicholphosphate. The enzyme is activated by the choline containing lipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. The dye Remazol blue acts as a competitive inhibitor of the enzyme with respect to UDP-Glc. The molecular weight of the enzyme has been determined to be approximately 37,000. The sole reaction product has been identified as dolichylphosphate glucose by isolation of the product by DEAE-cellulose chromatography and subsequent analysis of the acid-hydrolyzed product by both Bio-Gel P2 gel filtration and paper chromatography.     

Dolichyl phosphate phosphatase from Tetrahymena pyriformis.

A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.     

Purification and characterization of a third glucosyltransferase from Streptococcus mutans serotype g

Streptococcus mutans strain AHT (serotype g) secretes at least two glucosyltransferases with different pI values. A novel glucosyltransferase with a pI of 5.8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The Mr was estimated to be about 130,000 by SDS-PAGE and about 135,000 by ultracentrifugal analysis. The apparent Km value and pH optimum of the enzyme were 3.9 +/- 0.2 mM (mean +/- SD) and about 4.7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-alpha-D-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.     

Purification and properties of extracellular glucosyltransferase synthesizing 1,6-, 1,3-alpha-D-glucan from Streptococcus mutans serotype a

An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-alpha-D-glucan 3-alpha- and 6-alpha-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159 000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)-1 and the optimum pH was 6.0. The Km value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-alpha-linked glucose and 33.9 mol% 1,3-alpha-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-alpha-branched glucose.     

Functional expression of amylosucrase, a glucan-synthesizing enzyme, from Arthrobacter chlorophenolicus A6

A gene (acas) designated as alpha-amylase was cloned from Arthrobacter chlorophenolicus A6. The multiple amino acid sequence analysis and functional expression of acas revealed that this gene really encoded an amylosucrase (ASase) instead of alpha-amylase. In fact, the recombinant enzyme exhibited typical ASase activity by showing both sucrose hydrolysis and glucosyltransferase activities. The purified enzyme has a molecular mass of 72 kDa and exhibits optimal hydrolysis activity at 45 degrees C and a pH of 8.0. The analysis of the oligomeric state of ACAS with gel permeation chromatography revealed that the ACAS existed as a monomer.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

普鲁兰多糖高产菌株Y68葡萄糖基转移酶的纯化及特性研究

运用离子交换层析法和凝胶过滤层析法分离纯化普鲁兰多糖高产菌株Y68胞内葡萄糖基转移酶(简称GTF),并以SDS-PAGE、Native-PAGE对蛋白质进行量化、比较及特性鉴定,研究其酶学特性。结果表明短梗霉Y68中GTF被分离纯化,纯化酶在SDS-PAGE凝胶电泳上显示分子量50.8 ku单一条带,而在Native-PAGE凝胶电泳上显示分子量350 ku单一条带。纯化酶的最适酶反应pH和最适酶反应温度分别为6.0和40℃,酶对pH十分敏感,稳定性较差,对温度的稳定性则稍好。GTF为金属酶,Na+和K+对酶有激活作用,Ca2+、Mn2+、Mg2+、Ba2+、Cu2+、Fe2+、Hg2+、Co2+对酶活性有抑制作用,Hg2+对酶活性的抑制作用说明酶活性中心含有二锍键。EDTA、PMSF、SDS和碘乙酸对GTF活性有很大的抑制作用,而二硫苏糖醇(DTT)对酶活性有保护作用。     

Photoaffinity labeling of glucosyltransferase of the dolichol cycle from rat mammary gland

UDP-Glc:dolichol phosphate glucosyltransferase from lactating rat mammary gland has been partially purified by a combination of (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography on DEAE-TSK, and affinity chromatography. The partially purified enzyme exhibited several protein bands when examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; among these, a 35-kDa polypeptide was quite prominent and appeared to be enriched during purification. Photoaffinity labeling of the partially purified enzyme preparation with 5-azido-[beta-32P]UDP-Glc identified a 35-kDa polypeptide. Labeling of a solubilized enzyme preparation from crude and stripped microsomes also revealed a 35-kDa band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoinsertion of the probe in this polypeptide is enhanced by the presence of dolichol phosphate and Mg2+. Competition studies with UDP-Glc, UDP-glucuronic acid, other sugar nucleotides, and Glc-1-phosphate provide evidence to validate the specificity of photoaffinity labeling. These studies indicate that this 35-kDa polypeptide is involved in the synthesis of dolichol-P-Glc in rat mammary tissue. The possibility that this polypeptide may represent glucosyltransferase has been discussed.     

Solubilization and properties of UDP-D-glucose:N-acetylglucosaminyl pyrophosphorylundecaprenol glucosyltransferase from Bacillus coagulans AHU 1366 membranes

The glucosyltransferase which catalyzes the conversion of GlcNAc-PP-undecaprenol into Glc(beta 1----4)GlcNAc-PP-undecaprenol in the presence of UDP-glucose was solubilized from Bacillus coagulans AHU 1366 membranes by treatment with 0.1% Triton X-100 and partially purified by means of column chromatography on Sephacryl S-300 and DEAE-Sephacel. The final preparation was virtually free from other enzymes involved in the de novo synthesis of teichoic acid. The enzyme had a pH optimum of 6.6-8.0 and a Km value for UDP-glucose of 21 microM. The enzyme required 40 mM MgCl2, 0.6 M KCl, and 0.1% Nonidet P-40 for full activity.     

Changes in dehydrodolichyl diphosphate synthase during spermatogenesis in the rat

The levels of dolichyl phosphate and 2,3-dehydrodolichyl diphosphate synthase were determined in seminiferous tubules of prepuberal rats to assess any changes occurring during early stages of spermatogenesis. Dolichyl phosphate increased in concentration two- to threefold from Day 10 to Day 23 after birth. A method was optimized to measure dehydrodolichyl diphosphate synthesis from delta 3-[14C]isopentenyl diphosphate and t,t-farnesyl diphosphate in homogenates of seminiferous tubules. Both dehydrodolichyl mono- and diphosphates were observed as products of the in vitro assay. The specific activity of tubular synthase increased twofold between Day 7 and Day 23 and decreased similarly between Day 23 and Day 60. Since there was a parallel increase in the concentration of tubular dolichyl phosphate and dehydrodolichyl diphosphate synthase activity during early stages of spermatogenesis, it is proposed that the level of dolichyl phosphate may be controlled at least in part by the regulation of de novo dehydrodolichyl diphosphate biosynthesis. The synthase was also solubilized from tubular membranes with deoxycholate and partially purified by chromatography.     

Characteristics of glucosyltransferase from cultures ofStreptococcus mutans 6715 grown in trypticase soy broth and chemically defined media

Streptococcus mutants 6715 was grown in trypticase soy broth and chemically defined media. When compared by cellular mass, DNA content, acid production, or glucosyltransferase (GTase) production, the growth parameters were nearly identical. The doubling time for the organism grown in either medium was approximately 75 min. The extracellular glucosyltransferase produced byS. mutans 6715 grown in both media was purified from the culture supernatant with nearly total recovery and a degree of purification approximately 74-fold. Apparent proteolytic degradation of the enzyme was prevented by nitriloacetate. The temperature effects showed typical loss of enzymatic activity from 37° to 60°C. When the GTase was heated above 60°C there was partial restoration of activity. Immunological studies were used to establish the relationship between the enzymatically active proteins separated by gel filtration chromatography.     

Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A

Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic ribonuclease A (RNAseA enzyme) were solubilized from the rat liver microsomes in a non-ionic detergent, Triton X-100. Both enzyme activities were adsorbed on activated CH-Sepharose 4B, and could be eluted with a linear KCl gradient. Two enzyme activities were separated by this column with the dolichol enzyme eluting before the RNAseA enzyme. A 49-fold and 136-fold purification was achieved for the dolichol and the RNAseA enzyme, respectively. The addition of exogeneous dolichyl phosphate resulted in a 3-5-fold stimulation of the purified dolichol enzyme, but did not affect the purified RNAseA enzyme. The addition of RNAseA stimulated only the RNAseA enzyme. Whereas, tunicamycin could inhibit only the dolichol enzyme. The purified dolichol enzyme had a Km of 14 X 10(-6) M for UDP-GlcNAc and the reaction was saturated with about 0.25 M dolichyl phosphate. The purified RNAseA enzyme had a Km of 4.55 X 10(-6) M for UDP-GlcNAc and was saturated with about 0.36 mM RNAseA. The pH optima and the metal ion requirement for the two enzymes were different. These results suggest that because of the different properties of these two enzymes they may have distinct functions regarding the core glycosylation of N-linked glycoproteins. It is well established that the dolichol enzyme catalyzes the formation of the first dolichol-linked intermediate GlcNAc-P-P-dolichol, whereas according to the present finding, the RNAseA enzyme may catalyze the transfer of GlcNAc directly from UDP-GlcNAc into acceptor protein.     

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.     

Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids.

The activity of purified recombinant yeast dolichyl-phosphomannose synthase (EC 2.4.1.83) was assessed following reconstitution of the enzyme with phospholipids. The yeast synthase, similar to the mammalian enzyme, was active when reconstituted with phosphatidylethanolamine dispersions but had little (less than 5%) activity in stable phosphatidylcholine bilayers. The enzyme was activated by adding increasing amounts of diacylglycerol to phospholipid bilayers, suggesting that activity of the yeast enzyme was dependent on lipid phase properties rather than specific phospholipids. The synthase could also be reconstituted as an active enzyme in bilayers prepared with a commercial crude lipid preparation containing 40% phosphatidylcholine, but at a rate 10% of that occurring in phosphatidylethanolamine. Vesicles composed of the 40% phosphatidylcholine lipid mixture, dolichyl phosphate, and enzyme were leaky in the presence of divalent cations, and dolichyl-phosphomannose synthase did not appear to catalyze the translocation of dolichyl phosphomannose across membranes at a catalytically significant rate under the assay conditions employed.     

Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon.

A glucosyltransferase, which catalyzed the transfer of glucose from UDP-glucose (UDPG) to p-hydroxybenzoate (PHB) in cell cultures of Lithospermum erythrorhizon Sieb. et Zucc., Boraginaceae, was purified 219-fold by ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephadex G-150, and phenyl-Sepharose Cl-4B. p-Hydroxybenzoic acid O-beta-D-glucoside (PHB-glc) was identified as a product of the enzymatic reaction. This glucosyltransferase has a molecular weight of 47,500 Da, an isoelectric point at pH 5.0, and a pH optimum of 7.8. The enzyme does not sediment at 100,000g. Enzyme activity did not require metal cofactors. The enzyme was highly specific for p-hydroxybenzoate (Km 0.264 mM) and UDP-glucose (Km 0.268 mM). Initial velocity studies suggest that the enzyme reaction mechanism is a sequential rather than a ping-pong mechanism. Product inhibition patterns are consistent with an ordered sequential bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP the final product released. The data indicate the formation of a dead-end complex between PHB-glc and the enzyme. Uncompetitive inhibition by the substrate PHB can be put down to the formation of an abortive complex between E-UDP and PHB.