Leishmania donovani: regulated changes in the level of expression of the surface 3'-nucleotidase/nuclease

Leishmania donovani promastigotes have previously been shown to possess a surface membrane bound 3'-nucleotidase/nuclease (3'-N'ase) capable of hydrolyzing both nucleic acids and 3'-ribonucleotides. The specific activity of the 3'-N'ase was increased following transfer of the parasites to fresh, nutrient-replete media or to media lacking purines and/or inorganic phosphate (Pi). In nutrient-replete media, the enzyme activity was transiently elevated during the lag and early logarithmic phases of the growth curve; enzyme activity fell as the cells continued into late log and stationary phases. Purine- and Pi-starved cells exhibited significantly greater levels of 3'-N'ase activity than nutrient-replete cells. These levels remained elevated as long as the organisms were maintained in the deficient media. Nutrient-replete and purine-starved 125I surface-labeled parasites displayed differences in electrophoretic patterns. Upon purine starvation, incorporation of radiolabel was increased in proteins which migrated with apparent molecular weights of 70, 43, and 40 kDa. Comigration, in both one- and two-dimensional systems, of 3'-N'ase activity with the radiolabeled 43-kDa band demonstrated that this band was the catalytically active protein. Peptide mapping of the 70-, 43-, and 40-kDa proteins failed to demonstrate similarities in peptide sequence consistent with either a degradation or a precursor/product relationship. Treatment of the 43- and 40-kDa peptides with N-Glycanase indicated that they were differentially glycosylated. The cumulative results of these studies indicated that L. donovani can respond to altered culture conditions by the differential expression of surface proteins. In particular, the differential expression of the protein responsible for 3'-N'ase activity is consistent with the role of this enzyme in purine acquisition.     

Purification of Neuropathy Target Esterase from Avian Brain After Prelabelling with [3H]Diisopropyl Phosphorofluoridate

Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.     

Identification and characterization of variants of Crithidia luciliae with altered morphological and surface properties

Variants of a cloned laboratory stock of the trypanosomatid parasite Crithidia luciliae have been distinguished from "parental type" organisms. These variants accumulated spontaneously over time as the protozoan was maintained by continuous passage in a chemically defined medium. Cloned lines of these variants have been isolated by plating on nutrient agar and partially characterized on the basis of their growth characteristics in culture, their colony and cellular morphology as well as their surface protein expression. One cloned line consisted of motile, flagellated forms which, unlike "parental type" organisms, did not adhere to the surface of culture flasks. Another cloned line was composed of non-adherent, nonmotile, amastigote-like forms which were further distinguished from "parental type" cells by virtue of their constitutive expression, in nutrient-replete medium, of high levels of a surface membrane associated 3'-nucleotidase/nuclease (3'-N'ase) activity. Both the motile, flagellated and amastigote-like variants, like the "parental type" organisms, exhibited elevated levels of the 3'-N'ase activity upon exposure to purine starvation conditions. The variants described are of potential importance in elucidating the mechanism of induction of the highly regulated 3'-N'ase activity as well as for understanding the cytoskeletal systems and the surface properties of these protozoa.     

Identification and Characterization of Variants of Crithidia luciliae with Altered Morphological and Surface Properties

ABSTRACT. Variants of a cloned laboratory stock of the trypanosomatid parasite Crithidia luciliae have been distinguished from "parental type" organisms. These variants accumulated spontaneously over time as the protozoan was maintained by continuous passage in a chemically defined medium. Cloned lines of these variants have been isolated by plating on nutrient agar and partially characterized on the basis of their growth characteristics in culture, their colony and cellular morphology as well as their surface protein expression. One cloned line consisted of motile, flagellated forms which, unlike "parental type" organisms, did not adhere to the surface of culture flasks. Another cloned line was composed of non-adherent, nonmotile, amastigote-like forms which were further distinguished from "parental type" cells by virtue of their constitutive expression, in nutrient-replete medium, of high levels of a surface membrane associated 3'-nucleotidase/nuclease (3'-N'ase) activity. Both the motile, flagellated and amastigote-like variants, like the "parental type" organisms, exhibited elevated levels of the 3'-N'ase activity upon exposure to purine starvation conditions. The variants described are of potential importance in elucidating the mechanism of induction of the highly regulated 3'-N'ase activity as well as for understanding the cytoskeletal systems and the surface properties of these protozoa.     

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.     

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.     

Alterations in Leishmania donovani 3'-nucleotidase/nuclease activity banding pattern in polyacrylamide gels

1. In the absence of protease inhibitors, partial purification of the 3'-nucleotidase/nuclease (3'-N'ase) from detergent extracts of Leishmania donovani leads to the appearance of bands, at 42,000 and 38,000 Da, of enzyme activity which, in SDS-PAGE, migrate faster than 43,000 Da, the apparent weight of the intact polypeptide. 2. The generation of these faster migrating species is a consequence of detergent extraction and is prevented by the addition of the protease inhibitors PMSF and leupeptin, suggesting that degradation by an endogenous protease is responsible. 3. Treatment of leishmanial membranes with exogenous proteases yields activity at 38,000 Da which is membrane-associated. Protease treatment has no effect on living cells. 4. The observed changes in enzyme migration are discussed in terms of enzyme stability and regulation. 5. The advantages and limitations of the in situ gel activity assay are considered.     

Purification and properties of cyclic-3',5'-GMP-dependent protein kinase from the nematode Ascaris suum

A cyclic-3',5'-GMP-dependent protein kinase was purified 7400-fold from the reproductive tract of female ascarids to a specific activity of 718 nmol min-1 mg-1 (histone as substrate). The yield of the preparation was 25%. The enzyme protein obtained was homogeneous as judged by isoelectrofocusing and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme behaved as a dimer of two 82-kDa subunits in gel permeation chromatography on Superose 12. The protein kinase was inactive in the absence of cyclic purine nucleotides. Half-maximum velocity was obtained in the presence of 18 nM cGMP, whereas 400-fold higher concentrations of cAMP were required for the same activity. The enzyme underwent autophosphorylation in first-order kinetics (rate constant 0.054 min-1), leading to maximum incorporation of 0.96 phosphate per subunit. The autophosphorylation led to a 4-fold increase in Vmax, while the Km remained almost unchanged. In an extract from the reproductive tract, cGMP-stimulated phosphorylation was primarily observed in five proteins (molecular masses of 66, 60, 43, 30, and 25 kDa). These proteins also incorporated phosphate when isolated reproductive tracts were incubated in the presence of [32P]phosphate. The phosphate content in cellular proteins was enhanced when the incubation was performed in the presence of 10(-4) M of either octyl-cAMP or octyl-cGMP. In addition to the proteins mentioned above, however, six more electrophoretic bands containing radioactive phosphate were identified after in situ labeling of reproductive tracts with radioactive phosphate.     

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [3H]dexamethasone 21-mesylate ([3H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing, non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [3H]DM-labeled tryptic fragments resolved two (pI congruent to 5.7 and 7.0) 26.5-kDa and two (pI congruent equal to 5.7 and 6.8) 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [3H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [3H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments.     

Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.     

Studies on the regulation of 1-aminocyclopropane-1-carboxylate synthase in tomato using monoclonal antibodies

A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting <1 ng of antigen was developed. The ELISA system was used to demonstrate that two of the MAbs recognized different epitopes on the ACC-synthase protein. Wound-induced increases in ACC-synthase activity in tomato fruit tissue were correlated with changes in ELISA-detectable protein. In-vivo labeling of wounded tissue with [³⁵S]methionine followed by extraction and immunopurification in the presence of various protease inhibitors yielded one major radioactive band of 50 kDa molecular mass. Pulse labeling with [³⁵S]methionine at various times after wounding indicated that the wound-induced increase in ACC-synthase activity involved de-novo synthesis of a rapidly turning over 50-kDa polypeptide.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane

A phospholipase A2 activity directed against phosphatidylcholine was previously described in brush-border membrane from guinea pig intestine (Diagne, A., Mitjavila, S., Fauvel, J., Chap, H., and Douste-Blazy, L. (1987) Lipids 22, 33-40). In the present study, this enzyme was solubilized either with Triton X-100 or upon papain treatment, suggesting a structural similarity with other intestinal hydrolases such as leucine aminopeptidase, sucrase, or trehalase. The papain-solubilized form, which is thought to lack the short hydrophobic tail responsible for membrane anchoring, was purified 1800-fold to about 90% purity by ion exchange chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA44, and hydrophobic chromatography on phenyl-Sepharose. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a main band with an apparent molecular mass of 97 kDa was detected under reducing and nonreducing conditions. In the latter case, phospholipase A2 activity could be recovered from the gel and was shown to coincide with the 97-kDa protein detected by silver staining. The enzyme activity was unaffected by EGTA and slightly inhibited by CaCl2. The purified enzyme displayed a similar activity against phosphatidylcholine and phosphatidylethanolamine, whereas 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 50% compared to diacylglycerophospholipids. Using phosphatidylcholine labeled with either [3H]palmitic acid or [14C]linoleic acid in the 1- or 2-positions, respectively, the purified enzyme catalyzed the removal of [3H]palmitic acid, although at a lower rate compared to [14C]linoleic acid. This resulted in the formation of sn-glycero-3-phosphocholine, but only 1-[3H]palmitoyl-sn-glycero-3-phosphocholine was detected as an intermediary product. In agreement with this, 1-acyl-2-lyso-sn-[14C]glycero-3-phosphocholine was deacylated at almost the same rate as the sn-2-position of phosphatidylcholine. Since upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two hydrolytic activities were detected at the same position as 97-kDa protein, the enzyme is thus considered as a phospholipase A2 with lysophospholipase activity (phospholipase B), which might be involved in phospholipid digestion.     

Evidence for increased synthesis as well as increased degradation of protein kinase C after treatment of human osteosarcoma cells with phorbol ester

Phorbol 12-myristate 13-acetate (PMA) induces time-dependent changes in protein kinase C subcellular distribution and enzymatic activity in the human osteosarcoma cell line SaOS-2. Short (less than 60 min) incubations with PMA caused decreased cytosolic enzyme activity and a concomitant increase in particulate protein kinase; after 3 h, particulate protein kinase C activity also declined to reach less than 10% of basal activity by 24 h (Krug, E., and Tashjian, Jr., A. H., (1987) Cancer Res. 47, 2243-2246). In order to determine whether the loss in enzyme activity was due to decreased enzyme protein, Western blot analyses were performed using a polyclonal antibody against protein kinase C raised in rabbits. This approach confirmed the previously reported time-related changes: 80-kDa immunoreactive protein kinase C initially translocated from the cytosol to the particulate cell fraction and later disappeared completely from the particulate fraction. Loss of protein kinase C enzymatic activity thus results from actual loss of the 80-kDa protein; we found no evidence for generation of a calcium/phospholipid-independent protein kinase C-like form of the enzyme. Membrane association was confirmed by immunoprecipitation experiments using [35S]methionine-labeled cells. Brief exposure to PMA caused a marked loss in the [35S]methionine-labeled cytosolic protein kinase C band and an increase in the labeled particulate band. Protein kinase C immunoprecipitated from cells treated with PMA for 14 h displayed an increase in [35S]methionine label despite a greater than 80% loss of enzyme activity. The high specific radioactivity of the remaining 80-kDa protein leads us to conclude that long term treatment with PMA causes an increase in the rate of protein kinase C synthesis accompanied by a still greater increase in the rate of enzyme degradation in SaOS-2 cells.     

Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.     

Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

Studies were conducted to determine whether protein phosphorylation may be a mechanism for regulation of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS), shown previously to be light-dark regulated by some type of covalent modification. Radioactive phosphate was incorporated into the 120-kDa subunit of SPS during labeling of excised leaves with [32P]Pi, as shown by immunoprecipitation and denaturing gel electrophoresis of the enzyme. Conditions which activated the enzyme (illumination of leaves or mannose treatment of leaf discs in darkness) reduced the incorporation of radiolabel into SPS in the in vivo system. The partially purified SPS protein could also be phosphorylated in vitro using [gamma-32P]ATP. In the in vitro system, the incorporation of radiolabel into the 120-kDa subunit of SPS was dependent on time and magnesium concentration, and was closely paralleled by inactivation of the enzyme. These results provide the first evidence to establish protein phosphorylation as a mechanism for the covalent regulation of SPS activity.     

Biosynthesis of the adenosine deaminase-binding protein in human fibroblasts and hepatoma cells

The adenosine deaminase-binding protein has previously been localized to the cell surface of human fibroblasts (Andy, R. J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925). In this study we examine the biosynthesis of binding protein in human fibroblasts, human hepatoma HepG2 cells, and a human kidney tumor cell line. Binding protein immunoprecipitated from radioiodinated detergent-extracted fibroblast membranes has a molecular weight of 120,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional band of Mr 100,000 is also present which we believe is a result of proteolysis of the 120,000 band. Purified soluble kidney binding protein has an Mr of 112,000. Binding protein from fibroblasts pulse-labeled with [35S]methionine for 15 min migrates as a 110-kDa band on sodium dodecyl sulfate-polyacrylamide gels. Within 30-60 min of chase, the intensity of the 110-kDa band is diminished, and a 120-kDa band has appeared. Binding protein reaches the cell surface of fibroblasts within 30-60 min of chase. The same results are obtained with the other cell lines studied. Thus, binding protein is initially synthesized as a precursor of 110 kDa which chases into a 120-kDa mature form. The shift of 10 kDa is probably due to processing of its oligosaccharide chains since soluble kidney-binding protein contains 7-9 complex N-linked chains. Upon endoglycosidase H treatment, the 110,000 precursor shifts to a Mr of 89,000 while the 120,000 mature band shifts to 115,000, consistent with the presence of 7-9 high mannose chains on the precursor and 1-2 high mannose chains on the mature form. These results and the presence of complex N-linked chains on binding protein were confirmed by lectin affinity chromatography of glycopeptides derived from [2-3H]mannose-labeled binding protein. Analysis of [6-3H]glucosamine-labeled binding protein indicates the presence of 1 sialic acid residue per chain.     

Evidence for the Presence of a Free C-Terminal Fragment of Cx43 in Cultured Cells

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete® and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.     

Evidence for the presence of a free C-terminal fragment of cx43 in cultured cells

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete(R) and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.     

Purification, characterization, and subunit structure of rat core 1 Beta1,3-galactosyltransferase.

The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Galbeta1-->3GalNAcalpha1-->Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T). Core 1 beta3-Gal-T activity, which requires Mn2+, was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 beta3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of approximately 84- and approximately 86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of approximately 43 kDa. Reducing SDS-PAGE of these proteins gave approximately 42- and approximately 43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent Km for UDP-Gal of 630 microm and an apparent Vmax of 206 micromol/mg/h protein using GalNAcalpha1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment with O-glycosidase generated the expected disaccharide Galbeta1-->3GalNAc. These studies demonstrate that activity of the core 1 beta1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.     

Purification and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon,Thermoplasma acidophilum

We purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography. Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified protein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species. The gene encoding GGGP synthase in T. acidophilum was cloned after PCR amplification of the gene from the genomic DNA. The recombinant enzyme was expressed in Escherichia coli and purified. A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis. The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chromatography, suggesting that the enzyme is active as a homodimer. As the GGGP synthase from Methanobacterium thermoautotrophicum has been reported as a pentamer, the enzymes of the two organisms have different oligomeric structures. Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.