Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases

The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans. Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate. Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme. Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin. On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40. This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound. DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P. Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases.     

Application of 5-azido-UDP-glucose and 5-azido-UDP-glucuronic acid photoaffinity probes for the determination of the active site orientation of microsomal UDP-glucosyltransferases and UDP-glucuronosyltransferases.

A new approach to determining the active site orientation of microsomal glycosyltransferases is presented which utilizes the photoaffinity analogs [32P]5-Azido-UDP-glucose ([32P]5N3UDP-Glc) and [32P]5-Azido-UDP-glucuronic acid ([32P]5N3UDP-GlcA). It was previously shown that both photoprobes could be used to photolabel UDP-glucose:dolichol phosphate glucosyltransferase (Glc-P-Dol synthase), as well as the family of UDP-glucuronosyltransferases in rat liver microsomes. The effects of detergents, proteases, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the photolabeling of these enzymes were examined in intact rat liver microsomes. Photolabeling of Glc-P-Dol synthase by either photoprobe was the same in intact or disrupted vesicles, was susceptible to trypsin digestion, and was inhibited by the nonpenetrating inhibitor DIDS. Photolabeling of the UDP-glucuronosyltransferases by [32P]5N3UDP-GlcA was stimulated 1.3-fold in disrupted vesicles as compared to intact vesicles, whereas photolabeling of these enzymes by [32P]5N3UDP-Glc showed a 14-fold increase when vesicles were disrupted. Photolabeled UDP-glucuronosyltransferases were only susceptible to trypsin digestion in disrupted vesicles, and this was further verified by Western blot analyses. The results indicate a cytoplasmic orientation for access of UDP-sugars to Glc-P-Dol synthase and a lumenal orientation of most UDP-glucuronosyltransferases.     

Effect of anion-specific inhibitors on the utilization of sugar nucleotides for N-linked carbohydrate unit assembly by thyroid endoplasmic reticulum vesicles

The effect of anion-specific inhibitors on the utilization of the sugar nucleotides (UDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine) required for the formation of the oligosaccharide-lipid involved in N-glycosylation has been studied in intact endoplasmic reticulum (ER) vesicles from thyroid. Of the reagents tested, the nonpenetrating probe DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) and its dihydro derivative (H2DIDS) were the most effective, causing a pronounced impairment in the synthesis from UDP-Glc of dolichyl phosphate (Dol-P) glucose (50% reduction at 60 microM DIDS) and in the incorporation of glucose into oligosaccharide-lipid and N-glycosylated protein; in contrast, no inhibition was observed in the formation from UDP-Glc of a glycogen-like proteoglucan. The specificity of the DIDS effect was indicated by the finding that methyl isothiocyanate, a nonanionic amino-reactive agent, demonstrated negligible inhibition. While DIDS also effected a block in the formation of Dol-P-P-GlcNAc from UDP-GlcNAc, no impairment in the utilization of GDP-Man for Dol-P-Man synthesis was observed. Since the DIDS inhibition of UDP-Glc and UDP-GlcNAc utilization was maintained after disruption of the ER vesicles with Triton, even when the incubations were supplemented with Dol-P, it appears that this reagent does not interact with sugar nucleotide translocator proteins but rather with the cytoplasmically oriented anion binding sites of glycosyltransferases (UDP-Glc- and UDP-GlcNAc:Dol-P glucosyl- and GlcNAc-1-P transferases). This is consistent with the protease sensitivity of these enzymes in the intact ER vesicles. Incubation of the vesicles with tritiated H2DIDS (8 microM) introduced radioactivity into membrane polypeptides with molecular weights of about 52,000 and 31,000 as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this inhibitor may prove useful as an affinity label in further studies of some of the glycosyltransferases involved in the synthesis of lipid-monosaccharide intermediates.     

Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization

Metabolite glycosylation is affected by three classes of enzymes: nucleotidylyltransferases, which activate sugars as nucleotide diphospho-derivatives, intermediate sugar-modifying enzymes and glycosyltransferases, which transfer the final derivatized activated sugars to aglycon substrates. One of the first crystal structures of an enzyme responsible for the first step in this cascade, alpha-D-glucopyranosyl phosphate thymidylyltransferase (Ep) from Salmonella, in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 A and 2.1 A resolution, respectively. These structures, in conjunction with the kinetic characterization of Ep, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of Ep produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type Ep. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral component of in vitro glycosylation systems developed for the production of diverse glycorandomized libraries.     

Characterization of a protein:glucosyltransferase activity in human platelets

Human platelets exhibited significant glucosyltransferase activity, that transfer [14C]glucose from UDP-Glc to an endogenous protein acceptor. The enzyme protein:glucosyltransferase responsible for the catalysis was characterized and compared with glycogen:glucosyltransferase. We describe a partial separation of both activities, the ratio of protein:glucosyltransferase/glycogen:glucosyltransferase varied from 7:1 in a crude homogenate of platelets to 36:1 in the Sephadex G-100 column. This procedure failed to separate the protein:glucosyltransferase from its endogenous acceptor. Glucosylation of protein demonstrated dependence with respect to time and both protein and UDP-Glc concentration, and was saturated by very low concentration of donor and acceptor substrates. It was inhibited 76% by 5 mM Mn2+ concentration and was activated 23 and 11% by 5 mM concentrations of Ca2+ and Mg2+, respectively. With respect to glycogen:glucosyltransferase, when the effect of time, protein, and substrate concentration were determined under identical conditions, it did not show the same dependence. At 5 mM concentration, Mn2+, Ca2+, and Mg2+ were activators of the enzyme 43, 80, and 200%, respectively. On the basis of these characteristics, we conclude that the synthesis of glucoprotein and glycogen are catalyzed by two distinct enzymes. Addition of exogenous glycogen (range 0.002-1%) inhibited the protein:glucosyltransferase, whereas at 0.001-0.007% concentration it was acceptor substrate for glycogen:glucosyltransferase activity. At higher concentrations this activity was strongly inhibited. The concentration of glycogen in platelets could play a regulatory role in forming the glucoprotein and the glycogen molecules.     

Peroxisomal dihydroxyacetone phosphate acyltransferase. Effect of acetaldehyde on the intact and solubilized activity

The peroxisomal enzyme dihydroxyacetone phosphate (DHAP) acyltransferase shows a differential response to acetaldehyde. Employing whole peroxisomes, the enzyme displays a 130-400% stimulation of activity when assayed in the presence of 10-250 mM acetaldehyde. Following taurocholate solubilization of the enzyme the response to 0.25 M acetaldehyde is one of almost total inhibition. This inhibition of the taurocholate-solubilized enzyme is not observed at acetaldehyde concentrations below 200 mM. The stimulation of DHAP acyltransferase by acetaldehyde is solely a response of the peroxisomal enzyme as evidenced by its insensitivity to N-ethylmaleimide and 5 mM glycerol 3-phosphate. Furthermore, microsomal dihydroxyacetone phosphate acyltransferase activity is inhibited at all acetaldehyde concentrations. The activation of membrane-bound DHAP acyltransferase by acetaldehyde appears to be specific for this enzyme in comparison to several other peroxisomal and microsomal enzymes. The specificity of activation and differential response of the peroxisomal enzyme to acetaldehyde indicates that the microenvironment of the peroxisomal membrane is important for normal enzymatic function of this enzyme.     

Effect of bis-(p-nitrophenyl) phosphate on the biosynthesis and the utilization of lipid-intermediates.

Incubations of rat spleen lymphocytes with the required labelled nucleotide sugars lead to the formation of the various lipid-intermediates involved in the N-glycosylation of proteins. The effect of bis-(p-nitrophenyl) phosphate on the different reactions involved in the dolichol pathway has been studied. Although dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl diphosphate N-acetylglucosamine synthesis is not affected at all by bis-(p-nitrophenyl) phosphate (20 mM), this product inhibits completely the addition of the second N-acetylglucosamine residue on the dolichyl diphosphate N-acetylglucosamine acceptor. The addition of the five innermost mannose residues from GDP-mannose as donor is also strongly abolished. However, the addition of the more distal sugars, i.e. the four mannose residues using dolichyl phosphate mannose as donors and the additional glucose residues are only slightly affected. The reactions involved in the utilization of dolichyl diphosphate oligosaccharide, i.e. transfer to the proteins or degradation into soluble phospho-oligosaccharides, are also strongly inhibited. Thus bis-(p-nitrophenyl) phosphate appears to affect only the reactions involving the presence of dolichyl diphosphate sugar as substrate.     

Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets.

It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with bis-(p-nitrophenyl) phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards 5'-dTMP-P-nitrophenyl ester, which is probably associated with intracellular membrane structures in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.     

The UDP-Glc:glycoprotein glucosyltransferase is a soluble protein of the endoplasmic reticulum.

N-linked oligosaccharides devoid of glucose residues are transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum. The reaction products have been identified, depending on the organisms, as protein-linked Glc1Man5-9GlcNAc2. Incubation of right side-sealed vesicles from rat liver with UDP-[14C]Glc, Ca2+ ions and denatured thyroglobulin led to the glucosylation of the macromolecule only when the vesicles had been disrupted previously by sonication or by the addition of detergents to the glucosylation mixture. Similarly, maximal glucosylation of denatured thyroglobulin required disruption of microsomal vesicles isolated from the protozoan Crithidia fasciculata. Treatment of the rat liver vesicles with trypsin led to the inactivation of the UDP-Glc:glycoprotein glucosyltransferase only when proteolysis was performed in the presence of detergents. The glycoprotein glucosylating activity could be solubilized upon sonication of right side-sealed vesicles in an isotonic medium, upon passage of them through a French press or by suspending the vesicles in an hypotonic medium. Moreover, the enzyme appeared in the aqueous phase when the vesicles were submitted to a Triton X-114/water partition. Solubilization was not due to proteolysis of a membrane-bound enzyme. The enzyme could also be solubilized from C. fasciculata microsomal vesicles by procedures not involving membrane disassembly. About 30% of endogenous glycoproteins glucosylated upon incubation of intact rat liver microsomal vesicles with UDP-[14C]GLc could be solubilized by sonication or by suspending the vesicles in 0.1 M Na2CO3. These and previous results show that the UDP-Glc:glycoprotein glucosyltransferase is a soluble protein present in the lumen of the endoplasmic reticulum. In addition, both soluble and membrane-bound glycoproteins may be glucosylated by the glycoprotein glucosylating activity.     

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.     

The inhibition of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) dehydrochlorinase and gluthathione S-aryltransferase in grass-grub and housefly preparations

The inhibition of DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] dehydrochlorinase and glutathione S-aryltransferase by diphenylmethane and triphenylmethane derivatives was examined. Bis-(3,5-dibromo-4-hydroxyphenyl)methane and similar compounds were excellent inhibitors of both enzymes, but only DDT dehydrochlorinase was inhibited by compounds similar to bis-(N-dimethylaminophenyl)methane. Colour salts of the basic triphenylmethyl dyes were excellent inhibitors of both enzymes. All the inhibitors examined appeared to act by competition with glutathione for its binding site on the two enzymes.     

Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.     

Purification to apparent homogeneity and partial characterization of rat liver UDP-glucose:glycoprotein glucosyltransferase.

The UDP-Glc:glycoprotein glucosyltransferase is a soluble protein of the endoplasmic reticulum that catalyzes the glucosylation of protein-linked, glucose-free, high mannose-type oligosaccharides. In vivo, the newly glucosylated compounds are immediately deglucosylated, presumably by glucosidase II. The glucosyltransferase has been purified to apparent homogeneity from rat liver. The enzyme appears to have a molecular weight of 150,000 and 270,000 under denaturing and native conditions, respectively. The pure enzyme shows an almost absolute requirement for Ca2+ ions and for UDP-Glc as sugar donor. The same as crude preparations, the pure enzyme synthesized Glc1 Man7-9GlcNAc2-protein from Man7-9GlcNAc2-protein. Denatured glycoproteins are glucosylated much more efficiently than native ones by the apparently homogeneous glucosyltransferase. Availability of the pure enzyme will allow testing the possible involvement of transient glucosylation of glycoproteins in the folding of glycoproteins and/or in the mechanism by which cells dispose of malfolded glycoproteins in the endoplasmic reticulum.     

Rat intestinal phosphodiesterase II. Properties of the highly purified enzyme and its inactivation by iodoacetic acid.

A highly purifed preparation of rat intestinal phosphodiesterase II (oligonucleate 3'-nucleotidohydrolase, EC 3.1.4.18) has been studied using a synthetic substrate, thymidine 3'(2,4-dinitrophenyl) phosphate. The enzyme was most active between pH 6.1 and pH 6.7 and was inhibited by Cu2+ and Zn2+ but unaffected by EDTA, Mg2+, Co2+, and Ni2+. The reaction rate decreased at high levels of enzyme because of competitive inhibition by deoxythymidine 3'-phosphate, a reaction product, which showed a Ki of 2-10(-5) M. The molecular weight of the enzyme by gel-filtration was 150 000-170 000. In electrofocusing experiments multiple peaks of activity were found at pH 3.4, 4.2-4.5and 7.2. Polyacrylamide gel electrophoresis of freshly purified phosphodiesterase II showed up to 10 protein bands in the gels. If the preparations were stored at 4 degrees C for some time only one or two bands appeared. Investigation of the reaction of rat intestinal phosphodiesterase II with a number of possible phosphodiesterase substrates indicated that the enzyme required a nucleoside 3'-phosphoryl residue for the initiation of hydrolysis. Thus compounds such as NAD, ATP, bis-(p-nitrophenyl)phosphate, thymidine 5'-(p-nitrophenyl)phosphate, glycerylphosphorylcholine, guanylyl-(2' leads to 5')-adenosine and 3',5'-cyclic AMP which contain phosphodiester bonds, nevertheless were not substrates for the enzyme. The enzyme was inhibited reverisbly by p-chloromercuribenzoate and p-chloromercuriphenylsulfonate and inactivated irreversibly by iodoacetic acid. Activity of the phosphodiesterase II was reduced to 50% by incubation with 2.0-10(-3)--5.0-10(-3) M iodoacetate for 20--30 min at 24 degrees C at pH 5.0--6.1. Iodoacetamide had no effect. The degree of inactivation by iodoacetate was reduced by the presence of a substrate for the enzyme or, more effectively by deoxythymidine 3'-phosphate, a competitive inhibitor. It is concluded that iodoacetic acid alkylates an essential residue at the active centre of the enzyme.     

Steady state kinetics and effect of SH inhibitors on acid phosphatase from bovine brain.

1. Product inhibition studies and transphosphorylation to methanol using two different substrates indicate that acid phosphatase from bovine brain forms a phosphoryl enzyme and that the phosphorylation step can not be rate limiting. 2. Acid phosphatase from bovine brain is inhibited by 5,5'-dithiobis-(2-nitrobenzoic acid); this inhibition can be counteracted by inorganic phosphate. Incubation of the enzyme with p-nitrophenyl phosphate in the presence of p-chloromercuribenzoate leads, initially, to a higher degree of inhibition than that found with the same concentration of inhibitor in the absence of substrate. Both the titration by 5,5'-dithiobis-(2-nitrobenzoic acid) and inhibition by p-chloromercuribenzoate are consistant with the presence of 2 SH groups per mol of enzyme.     

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.     

GDP-fucose uptake into the Golgi apparatus during xyloglucan biosynthesis requires the activity of a transporter-like protein other than the UDP-glucose transporter

The molecular mechanisms regulating hemicelluloses and pectin biosynthesis are poorly understood. An important question in this regard is how glycosyltransferases are oriented in the Golgi cisternae, and how nucleotide sugars are made available for the synthesis of the polymers. Here we show that the branching enzyme xyloglucan alpha,1-2 fucosyltransferase (XG-FucTase) from growing pea (Pisum sativum) epicotyls was latent and protected against proteolytic inactivation on intact, right-side-in pea stem Golgi vesicles. Moreover, much of the XG-FucTase activity was membrane associated. These data indicate that XG-FucTase is a membrane-bound luminal enzyme. GDP-Fuc uptake studies demonstrated that GDP-Fuc was taken up into Golgi vesicles in a protein-mediated process, and that this uptake was not competed by UDP-Glc, suggesting that a specific GDP-Fuc transporter is involved in xyloglucan biosynthesis. Once in the lumen, Fuc was transferred onto endogenous acceptors, including xyloglucan. GDPase activity was detected in the lumen of the vesicles, suggesting than the GDP produced upon transfer of Fuc was hydrolyzed to GMP and inorganic phosphate. We suggest than the GDP-Fuc transporter and GDPase may be regulators of xyloglucan fucosylation in the Golgi apparatus from pea epicotyls.     

The 5′-Nucleotidases and Cyclic Phosphodiesterases (3′-Nucleotidases) of the Enterobacteriaceae

All members of the Enterobacteriaceae possess distinct 5'-nucleotidases and cyclic phosphodiesterases (3'-nucleotidases) that can be differentiated from the acid and alkaline phosphatases and the acid sugar hydrolases. The nucleotidases and cyclic phosphodiesterases of the various Enterobacteriaceae are remarkably similar in properties. All of the 5'-nucleotidases hydrolyze 5'-nucleotides, adenosine triphosphate, and uridine diphosphoglucose. Their pH optimum is from 5.7 to 6.1. The cyclic phosphodiesterases hydrolyze 3'-nucleotides, cyclic phosphonucleotides, bis-(p-nitrophenyl)phosphate, and p-nitrophenylphosphate. Their pH optimum is from 7.2 to 7.8. For both enzymes, cobalt showed optimal metal stimulation. An intracellular protein inhibitor for the 5'-nucleotidase is present in all of the Enterobacteriaceae. No inhibitor of cyclic phosphodiesterase activity exists, although hydrolysis of both cyclic phosphonucleotides and 3'-nucleotides is inhibited by ribonucleic acid. Neither of the enzymes is subject to control by phosphate level or by catabolite repression. Of the other bacteria studied, only Haemophilus and Bacillus subtilis contained significant 3'- or 5'-nucleotidase activity.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

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.