Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene.

A gene encoding an alpha-1,2-mannosyltransferase from Saccharomyces cerevisiae was cloned and sequenced. The alpha-1,2-mannosyltransferase which utilizes alpha-methylmannoside as acceptor of mannose from GDP-mannose was purified. The enzyme activity was shown to correspond to a 41 kDa protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This protein band was digested in situ with trypsin and amino acid sequence information was obtained from four peptides. Degenerate oligonucleotide primers corresponding to the amino acid sequences were designed and used for polymerase chain reactions on yeast genomic DNA. A specific reaction product was used to screen a genomic library of S.cerevisiae. A fragment of approximately 5.7 kb was isolated, of which a 2.9 kb fragment was sequenced. It contained a 1329 base pair open reading frame encoding the peptide sequences of the purified alpha-1,2-mannosyltransferase. The gene, designated MNT1, is located on the right arm of chromosome 4. It encodes a 442 amino acid polypeptide with a calculated mol. wt of 51.4 kDa. The corresponding mRNA has a length of approximately 1.6 kb. Overexpression of the MNT1 gene increased this alpha-1,2-mannosyltransferase activity approximately 2.5-fold. The protein was shown to be modified with N-linked carbohydrate chains and its sequence contains one N-glycosylation site. The enzyme contains a putative membrane-spanning domain near its N-terminus and its topology is thus similar to that of mammalian Golgi glycosyltransferases. This is the first report of the cloning and sequencing of a yeast Golgi mannosyltransferase.     

Purification of gamma-glutamyltransferase by phenyl boronate affinity chromatography. Studies on the acceptor specificity of transpeptidation by rat kidney gamma-glutamyltransferase

gamma-Glutamyltransferase has been purified from rat kidney by a novel procedure using phenyl boronate affinity chromatography. The highly purified enzyme has been studied with respect to acceptor specificity for a number of amino acids, amino acid analogues, dipeptides and tripeptides. The acceptor activity is specific for L-amino acids. The amino acids and the majority of the essential amino acids are poor acceptors while the sulphur-containing amino acids are the best acceptors. The acceptor activity is modulated by the substitution of the amino acid side chain. Substitution of the side chain at the delta, gamma or beta positions results in a proportionally decreasing ability to act as acceptor. The carbonyl moiety of the gamma-carboxy group of the acceptor appears to be essential for acceptor activity, absence of an alpha-carboxy carbonyl group increases the Kappm of the acceptor approximately 100-fold.     

Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties.

gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.     

Cis and trans factors involved in apicoplast targeting in Toxoplasma gondii

Ribosomal subunit protein 9 (rps9) is a nuclearly encoded protein that resides in the apicoplast organelle of Toxoplasma gondii. Two cis-acting regions within the rps9 transit domain (amino acids 38-49 and 79-86), when combined with the rps9 signal sequence, were necessary and sufficient for apicoplast targeting. To investigate proteins interacting with the rps9 leader sequence, parasites expressing rps9 leader constructs fused to a glutathione S-transferase (GST) reporter were prepared, and proteins associated with the leader constructs were purified from extracts by affinity chromatography. In addition to GST-containing peptides, proteins with apparent masses of 92, 90, 86, and 160 kDa were purified. Mass spectrometry data suggested that the 92- and 90-kDa polypeptides appear to be subtilisin-like proteins, whereas the 86-kDa polypeptide was identified as the molecular chaperone BiP of T. gondii.     

Purification of GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase from Saccharomyces cerevisiae

The enzyme GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase (GDP-Man:DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP in equilibrium DolP-Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis. The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels. Enzymatic activity could be correlated directly with this band. Antibodies against the transferase were raised in rabbits. The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30-kDa band on immunoblots. Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N-ethylmaleimide treatment.     

Cloning, structural analysis, and expression of the glycogen phosphorylase-2 gene in Dictyostelium.

The glycogen phosphorylase-2 (GP2) activity that appears during the cell differentiation of Dictyostelium was purified to homogeneity. The molecular weight of the nondenatured enzyme was 200,000 as determined by Sephacryl S-300 gel filtration and was 107,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme consists of two similar subunits. The intact protein was digested with trypsin and protease V8, and the resulting peptides were purified by microbore high pressure liquid chromatography. The peptides were sequenced, and oligonucleotides were constructed for polymerase chain reaction amplification of the GP2 gene from Dictyostelium genomic DNA template. The resulting polymerase chain reaction products were sequenced directly and were confirmed to encode portions of the GP2 gene. These fragments were used to probe a partial EcoRI genomic library for the remainder of the GP2 gene. The nucleotide sequence of the GP2-selected clones revealed an open reading frame of 2975 base pairs that was interrupted by two introns of 109 and 105 base pairs, respectively. The open reading frame encoded a protein of 992 amino acids with a calculated molecular mass of 112,500 Da and an isoelectric point of 6.4. An unusual sequence within the second exon of GP2, in which the triplet CAA was repeated 11 times, resulted in 11 in-frame glutamine residues of a possible 15 amino acids coded for by this region. The CAA repeat was transcribed, as shown by the sequence of cDNA. Comparison of the amino acid sequence of Dictyostelium GP2 to the phosphorylases from other organisms revealed that the Dictyostelium protein was 50 and 44% identical to yeast and rabbit muscle phosphorylases, respectively. Northern blot analysis showed that GP2 mRNA was absent in amebas and the early stages of development, reached a maximum level of expression at the slug stage, and then decreased in the terminal stages of development. Comparison of the mRNA expression with the appearance of GP2 enzyme protein and enzyme activity revealed that gp2 mRNA and a 113-kDa GP2 enzyme peptide were expressed concurrently at 10 h of development. However, enzyme activity did not appear until 18 h, coincident with a decrease in the level of the 113-kDa peptide and a corresponding increase in the amount of a 106-kDa GP2 peptide. Addition of cAMP to aggregation-competent cells in liquid culture resulted in the induction of GP2 mRNA, GP2 protein, and GP2 enzyme activity.     

Characterization of mycobacterial protein glycosyltransferase activity using synthetic peptide acceptors in a cell-free assay

Synthetic peptides derived from a 45-kDa glycoprotein antigen of Mycobacterium tuberculosis were shown to function as glycosyltransferase acceptors for mannose residues in a mannosyltransferase cell-free assay. The mannosyltransferase activity was localized within both isolated membranes and a P60 cell wall fraction prepared from the rapidly growing mycobacterial strain, Mycobacterium smegmatis. Incorporation of radiolabel from GDP-[(14)C]mannose was inhibited by the addition of amphomycin, indicating that the glycosyl donor for the peptide acceptors was a member of the mycobacterial polyprenol-P-mannose (PPM) family of activated glycosyl donors. Furthermore, a direct demonstration of transfer from the in situ generated PP[(14)C]Ms was also demonstrated. It was also found that the enzyme activity was sensitive to changes in overall peptide length and amino acid composition. Because glycoproteins are present on the mycobacterial cell surface and are available for interaction with host cells during infection, protein glycosyltransferases may provide novel drug targets. The development of a cell-free mannosyltransferase assay will now facilitate the cloning and biochemical characterisation of the relevant enzymes from M. tuberculosis.     

Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.     

Arylamine N-acetyltransferase from chicken liver. I. Monoclonal antibodies, immunoaffinity purification, and amino acid sequences

Five monoclonal antibodies against arylamine acetyltransferase (EC 2.3.1.5) from the chicken liver were established by immunizing a mouse with a partially purified enzyme preparation. None of the antibodies cross-reacted with arylamine N-acetyltransferase from the livers of cow, rabbit, and rat, nor with arylalkylamine N-acetyltransferase from the chicken pineal gland, indicating a high specificity of the antibodies. By using the antibodies, two immunoaffinity purification procedures were elaborated: A partially purified enzyme preparation was incubated with the monoclonal antibody, and the resulting enzyme-IgG complex was separated by a protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular mass of 34 kDa in addition to the heavy and light chains of IgG. Secondly, an immunoaffinity column was prepared by immobilizing a monoclonal antibody to Sepharose 4B. After a partially purified enzyme preparation was absorbed on the column, N-acetyltransferase activity was eluted with 1 M NaCl and 1 M urea. The eluted sample contained a single 34-kDa protein. The purified enzyme preferred arylamines to arylalkylamines as substrates, indicating that it was arylamine N-acetyltransferase. The purified protein was subjected to digestion by lysylendopeptidase and separated by high performance liquid chromatography. Partial amino acid sequences of three peptides were determined by a gas-phase sequence analyzer.     

The proteolytic system of Lactobacillus sanfrancisco CB1: purification and characterization of a proteinase, a dipeptidase, and an aminopeptidase.

A cell envelope 57-kDa proteinase, a cytoplasmic 65-kDa dipeptidase, and a 75-kDa aminopeptidase were purified from Lactobacillus sanfrancisco CB1 sourdough lactic acid bacterium by sequential fast protein liquid chromatography steps. All of the enzymes are monomers. The proteinase was most active at pH 7.0 and 40 degrees C, while aminopeptidase and dipeptidase had optima at pH 7.5 and 30 to 35 degrees C. Relatively high activities were observed at the pH and temperature of the sourdough fermentation. The proteinase is a serine enzyme. Urea-polyacrylamide gel electrophoresis of digest of alpha s1- and beta-caseins showed differences in the pattern of peptides released by the purified proteinase and those produced by crude preparations of the cell envelope proteinases of Lactobacillus delbrueckii subsp. bulgaricus B397 and Lactococcus lactis subsp. lactis SK11. Reversed-phase fast protein liquid chromatography of gliadin digests showed a more-complex peptide pattern produced by the proteinase of Lactobacillus sanfrancisco CB1. The dipeptidase is a metalloenzyme with high affinity for dipeptides containing hydrophobic amino acids but had no activity on tripeptides or larger peptides. The aminopeptidase was also inhibited by metal-chelating agents, and showed a broad N-terminal hydrolytic activity including di- and tripeptides. Km values of 0.70 and 0.44 mM were determined for the dipeptidase on Leu-Leu and the aminopeptidase on Leu-p-nitroanilide, respectively.     

Glutamate-glyoxylate aminotransferase in rat liver cytosol. Purification, properties and identity with alanine-2-oxoglutarate aminotransferase

After cortisone injection, virtually identical increases in rat liver cytosol alanine-2-oxoglutarate aminotransferase and glutamate-glyoxylate aminotransferase activities were observed. The two activities were co-purified to homogeneity from rat liver cytosol. The purified enzyme was specific for L-alanine with 2-oxoglutarate as amino acceptor. With glyoxylate, however, the enzyme utilized various L-amino acids as amino donors in the following order of activity: glutamate greater than alanine greater than glutamine greater than methionine. The ratio of alanine-2-oxoglutarate aminotransferase activity to glutamate-glyoxylate aminotransferase activity remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. These results suggest that glutamate-glyoxylate aminotransferase is identical with alanine-2-oxoglutarate aminotransferase. Evidence was obtained that the two enzyme activities in the cytosol of dog, cat and human liver are also properties of the same protein.     

Alpha-enolase is restricted to basal cells of stratified squamous epithelium.

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.     

Aspartate: 2-oxoglutarate aminotransferase from trichomonas vaginalis. Identity of aspartate aminotransferase and aromatic amino acid aminotransferase.

Aspartate: 2-oxoglutarate aminotransferase from the anaerobic protozoon Trichomonas vaginalis was purified to homogeneity and characterized. It is a dimeric protein of overall Mr approx. 100000. Only a single isoenzyme was found in T. vaginalis. The overall molecular and catalytic properties have features in common with both the vertebrate cytoplasmic and mitochondrial isoenzymes. The purified aspartate aminotransferase from T. vaginalis showed very high rates of activity with aromatic amino acids as donors and 2-oxoglutarate as acceptor. This broad-spectrum activity was restricted to aromatic amino acids and aromatic 2-oxo acids, and no significant activity was seen with other common amino acids, other than with the substrates and products of the aspartate: 2-oxoglutarate aminotransferase reaction. Co-purification and co-inhibition, by the irreversible inhibitor gostatin, of the aromatic amino acid aminotransferase and aspartate aminotransferase activities, in conjunction with competitive substrate experiments, strongly suggest that a single enzyme is responsible for both activities. Such high rates of aromatic amino acid aminotransferase activity have not been reported before in eukaryotic aspartate aminotransferase.     

Cloning of a novel collagenase gene from the gram-negative bacterium Grimontia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis

The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif. G. hollisae collagenase showed 60 and 59% amino acid sequence identities to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In contrast, this enzyme showed < 20% sequence identity with Clostridium histolyticum collagenase. When the recombinant mature collagenase, which consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was produced by the Brevibacillus expression system, a major gelatinolytic protein band of ~ 60 kDa was determined by zymographic analysis. This result suggested that cloned collagenase might undergo processing after secretion. Moreover, the purified recombinant enzyme was shown to possess a specific activity of 5,314 U/mg, an ~ 4-fold greater activity than that of C. histolyticum collagenase.     

A peptidase activity exhibited by human serum pseudocholinesterase

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.     

A variant of arrestin-1 binds rod outer segment membranes in a light-independent manner

A 50-kDa-polypeptide band peripherally bound to retinal rod outer segment (ROS) membranes was purified by anion-exchange chromatography. When the 50-kDa protein was compared with purified arrestin-1, it was observed that: (1) both proteins comigrated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and were recognized by either anti-50-kDa protein polyclonal antibodies or anti-arrestin-1 monoclonal antibodies; (2) protein fragments and peptide fingerprint maps obtained following limited and complete proteolysis with specific proteases were very similar for both molecules; and (3) several chromatographically-purified tryptic peptides from the 50-kDa protein possessed the same amino acid composition as tryptic peptides deduced from the reported arrestin-1 primary structure. Consequently, arrestin-1 and the purified 50-kDa protein must correspond to variants of the same molecule. However, in contrast to arrestin-1 that associated to the ROS membranes only in the presence of light and ATP, the 50-kDa protein interacted with the ROS membranes in a light-independent manner, either in the presence or absence of ATP. These results clearly established that phosphorylated and illuminated rhodopsin is not the membrane anchor for this variant of arrestin-1.     

Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.     

Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.     

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.