Some properties of purified phosphoprotein phosphatases from rabbit liver.

The activity of two purified homogeneous phosphoprotein phosphatases types P I and P II) (phosphoprotein phosphohydrolase, EC 3.1.3.16) from rabbit liver (Khandelwal, R.L., Vandenheede, J.R., and Krebs, E.G. (1976) J. Biol. Chem. 251, 4850-4858) were examined in the presence of divalent cations, Pi, PPi, nucleotides, glycolytic intermediates and a number of other compounds using phosphorylase a, glycogen synthase D and phosphorylated histone as substrates. Enzyme activities were usually inhibited by divalent cations with all substrates; the inhibition being more pronounced with phosphorylase a. Zn2+ was the most potent inhibitor among the divalent cations tested. The enzyme was competitively inhibited by PPi (Ki = 0.1 mM for P I and 0.3 mM for PII), Pi (Ki = 15 mM for P I and 19.8 mM for P II) and p-nitrophenyl phosphate (Ki = 1 mM and 1.4 mM for P I and P II, respectively) employing phosphorylase a as the substrate. The compounds along with a number of others (Na2SO4, citrate, NaF and EDTA) also inhibited the enzyme activity with the other two substrates. Severe inhibition of the enzyme was also observed in the presence of the adenine and uridine nucleotides; monophosphate nucleotides being more inhibitory with phosphorylase a, whereas the di- and triphosphate nucleotides showed more inhibition with glycogen synthase D and phosphorylated histone. Cyclic AMP had no significant effect on enzyme activity with all the substrates tested. Phosphorylated metabolites did not show any marked effect on the enzyme activity with phosphorylase a as the substrate.     

Identification of a rat liver alpha-N-acetylglucosaminyl phosphodiesterase capable of removing "blocking" alpha-N-acetylglucosamine residues from phosphorylated high mannose oligosaccharides of lysosomal enzymes

We recently reported that the high mannose-type oligosaccharides of the biosynthetic intermediates of beta-glucuronidase contain phosphate groups in diester linkage between mannose residues and outer alpha-linked N-acetylglucosamine residues (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We now describe an alpha-N-acetylglucosaminyl phosphodiesterase from rat liver that is capable of removing the N-acetyl-glucosamine residues, leaving phosphomonoester groups on the high mannose oligosaccharide units. This activity is greatly enriched in smooth membrane preparations. It can be distinguished from a lysosomal alpha-N-acetylglucosaminidase by several criteria, including subcellular localization and differential inhibition by amino sugars. In addition, human fibroblasts with mutations which lead to a deficiency of the lysosomal activity have normal levels of the alpha-N-acetylglucosaminyl phosphodiesterase. This enzyme may be involved in the "unmasking" of the phosphomannosyl recognition marker on newly synthesized acid hydrolases which could then direct the targeting of these enzymes to lysosomes.     

Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2-terminal binding sites on a single enzyme

Previous studies have indicated the existence of separate binding sites of ubiquitin-protein ligase, E3, specific for basic (Type I) or bulky hydrophobic (Type II) NH2-terminal amino acid residues of proteins. Another class (Type III) of protein substrates appeared to interact with E3 at regions other than the NH2 terminus (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698). In the present study we have used affinity chromatography on immobilized protein substrates to examine the question of whether the different binding sites belong to one E3 enzyme, or to different E3 species. Another objective was to develop a procedure for the extensive purification of E3. When a crude extract of reticulocytes is applied to Type I or Type II protein substrates linked to Sepharose, E3 becomes strongly bound to the affinity columns and is not eluted with salt at high concentration. However, the enzyme can be specifically eluted by a dipeptide that has an NH2-terminal residue similar to that of matrix-bound protein substrate. A 350-fold purification is obtained in this single step. Preparations of E3 purified on either Type I or Type II protein substrate affinity columns act on both types of protein substrates, indicating that the separate binding sites for basic and hydrophobic NH2-terminal residues belong to one enzyme. Another species of E3 that acts strongly on some Type III protein substrates does not bind to Type I or Type II protein substrate affinity columns.     

Processing of the phosphorylated recognition marker in lysosomal enzymes. Characterization and partial purification of a microsomal alpha-N-acetylglucosaminyl phosphodiesterase

N-Acetylglucosamine(1)phospho(6)mannose groups recently identified in lysosomal enzymes were proposed to be precursors of the recognition markers terminating with mannose 6-phosphate (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 225, 6633-6639; Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura, K. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7074-7078). To study the presumptive enzyme removing N-acetylglucosamine from the diester, an assay was developed using a radioactive oligosaccharide containing diester groups of the above structure. An alpha-N-acetylglucosaminyl phosphodiesterase cleaving this substrate in vitro was found in human placenta and in rat liver. The enzyme was solubilized from the microsomal fraction of human placenta and more than 800-fold purified with 75% yield. It is distinct from the lysosomal alpha-N-acetylglucosaminidase by the criteria of immunological cross-reactivity, substrate specificity, and heat stability. The partially purified enzyme cleaves alpha-N-acetylglucosamine phosphodiester bonds in oligosaccharides from lysosomal enzymes, in lysosomal enzymes, and in UDP-N-acetylglucosamine. We propose that the microsomal alpha-N-acetylglucosaminyl phosphodiesterase is involved in the processing of the phosphorylated recognition marker in lysosomal enzymes.     

Conversion of endoglycoceramidase-activator II by trypsin to the 27.9 kDa polypeptide possessing full activity: purification of activator for endoglycoceramidase by trypsin treatment followed by trypsin-inhibitor agarose column application.

Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids [Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282]. A detergent was required for EGCase to express full activity, possibly due to its hydrophobic nature. Recently, activator proteins responsible for stimulating EGCase activity in the absence of detergents were isolated from the culture supernatant of Rhodococcus sp. [Ito, M., Ikegami, Y., & Yamagata, T. (1991) J. Biol. Chem. 266, 7919-7926]. The activity of activator II specific for EGCase II was heat-labile but insensitive to trypsin-treatment. This activator (69.2 kDa) was converted to the 27.9 kDa polypeptide via the 42 kDa intermediate by exhaustive trypsination, and the stimulatory activity of 27.9 kDa polypeptide on EGCase II was identical to that of the native form toward asialo GM1 and cell-surface GM3 of horse erythrocytes as substrates. This observation was successfully applied to obtain the purified activator without contamination with EGCase activity, which is abolished completely following treatment with trypsin.     

Ca2+-independent activation of protease-activated kinase II by phospholipids/diolein and comparison with the Ca2+/phospholipid-dependent protein kinase

The proenzyme form of protease-activated kinase (PAK) II from reticulocytes has been shown to be activated in vitro by limited proteolysis and characterized using 40 S ribosomal subunits as substrate (T.H. Lubben and J.A. Traugh (1983) J. Biol. Chem. 258, 13992-13997). In these studies, we have shown that PAK II can be activated in a Ca2+-independent manner with phospholipids/diolein using histone 1, eukaryotic initiation factor 2, and 40 S ribosomal subunits as substrates. The addition of Ca2+ results in a diminution of PAK II activity. The Ca2+/phospholipid-dependent protein kinase (protein kinase C) is present in reticulocytes and is separated from PAK II during purification by chromatography on ADP-agarose. PAK II activated by limited proteolysis has the same substrate specificity as PAK II activated by phospholipids/diolein as shown by two-dimensional finger-printing of tryptic phosphopeptides of histone 1 and ribosomal protein S6, indicating proteolysis did not alter the specificity of the enzyme. Lipid vesicles decrease the Km of PAK II for histone 1 by 10-fold, while no effect is observed on the Km or the Vmax of PAK II for ATP. These results are strikingly different from the kinetics reported for protein kinase C, where the activators increase the Vmax for ATP. The two enzymes have similar, if not identical, substrate specificity with histone 1, as determined by phosphopeptide mapping, but at least 8-fold more protein kinase C than PAK II is required to incorporate a comparable amount of phosphate into S6 and it is not possible to incorporate stoichiometric amounts of phosphate into S6 with protein kinase C. The two protein kinases also differentially phosphorylate other substrates. The data support the hypothesis that PAK II and protein kinase C are closely related, but unique enzymes.     

Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.     

Cellulases of Penicillium verruculosum

Nine major cellulolytic enzymes were isolated from a culture broth of a mutant strain of the fungus Penicillium verruculosum: five endo-1, 4-β-glucanases (EGs) having molecular masses 25, 33, 39, 52, and 70 kDa, and four cellobiohydrolases (CBHs: 50, 55, 60, and 66 kDa). Based on amino acid similarities of short sequenced fragments and peptide mass fingerprinting, the isolated enzymes were preliminary classified into different families of glycoside hydrolases: Cel5A (EG IIa, 39 kDa), Cel5B (EG IIb, 33 kDa), Cel6A (CBH II, two forms: 50 and 60 kDa), Cel7A (CBH I: 55 and 66 kDa), Cel7B (EG I: 52 and 70 kDa). The 25 kDa enzyme was identical to the previously isolated Cel12A (EG III). The family assignment was further confirmed by the studies of the substrate specificity of the purified enzymes. High-molecular-weight forms of the Cel6A, Cel7A, and Cel7B were found to possess a cellulose-binding module (CBM), while the catalytically active low-molecular-weight forms of the enzymes, as well as other cellulases, lacked the CBM. Properties of the isolated enzymes, such as substrate specificity toward different polysaccharides and synthetic glycosides, effect of pH and temperature on the enzyme activity and stability, adsorption on Avicel cellulose and kinetics of its hydrolysis, were investigated.     

Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues.

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.     

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris

The giant extracellular hemoglobin (3,800 kDa) of the oligochaete Lumbricus terrestris consists of four subunits: a monomer (chain I), two subunits each of about 35 kDa (chains V and VI), and a disulfide-bonded trimer (50 kDa) of chains II, III, and IV. The complete amino acid sequence of chain I was determined: it consists of 142 amino acid residues and has a molecular weight of 16,750 including a heme group. Fifty-nine residues (42%) were found to be identical with those in the corresponding positions in Lumbricus chain II (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015); 45 (32%), 56 (40%), 44 (31%), and 45 (32%) residues were found to be in identical positions in the sequences of chains I, IIA, IIB, and IIC, respectively, of Tylorrhynchus heterochaetus hemoglobin (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). When the sequences of all six annelid chains are compared, 18 invariant residues are found in the first 104 residues of the molecule; very little homology exists among the annelid chains in the carboxyl-terminal 38-residue region. Nine of the 18 invariant residues are also found in the human beta-globin chain.     

Rat intestinal nucleotide-sugar pyrophosphatase. Localization, partial purification, and substrate specificity

The nucleotide-sugar pyrophosphatase activity of rat small intestine was studied using GDP-[14C]Man as substrate. The highest specific activities in the gastrointestinal tract were in the proximal small intestine, with a preferential localization in villus tip cells. Purified brush-border membranes were highly enriched in nucleotide-sugar pyrophosphatase. After the enzyme was solubilized with detergent and purified 180-fold, it hydrolyzed FAD and p-nitrophenyl-5'-thymidylate, as well as nucleotide sugars. That the same enzyme, a 5'-nucleotide phosphodiesterase, is responsible for nucleotide-sugar pyrophosphatase, phosphodiesterase I, and FAD pyrophosphatase activities is indicated by: co-migration in electrophoresis, parallel thermal inactivation, competitive inhibition studies, and similar regional, cellular, and subcellular localizations.     

Isolation and preliminary characterization of two forms of ribulose 1,5-bisphosphate carboxylase from Rhodopseudomonas capsulata.

The presence of two distinct forms of ribulose 1,5-bisphosphate carboxylase has been demonstrated in extracts of Rhodopseudomonas capsulata, similar to the form I (peak I) and form II (peak II) carboxylases previously described from R. sphaeroides (J. Gibson and F. R. Tabita, J. Biol. Chem 252:943-949, 1977). The two activities, separated by diethylaminoethyl-cellulose chromatography, were shown to be of different molecular size after assay on polyacrylamide gels. The higher-molecular-weight carboxylase from R. capsulata was designated form I-C, whereas the smaller enzyme was designated form II-C. Catalytic studies revealed significant differences between the two enzymes in response to pH and the effector 6-phosphogluconate. Immunological studies with antisera directed against the carboxylases from R. sphaeroides demonstrated antigenic differences between the two R. capsulata enzymes; cross-reactivity was observed only between R. sphaeroides anti-form II serum and the corresponding R. capsulata enzyme, form II-C.     

Catalytic and regulatory properties of two forms of bovine heart cyclic nucleotide phosphodiesterase.

The soluble supernatant fraction of bovine heart homogenates may be fractionated on a DEAE cellulose column into two cyclic nucleotide phosphodiesterases (EC 3.1.4.-):PI and PII phosphodiesterases, in the order of emergence from the column. In the presence of free Ca2+, the PI enzyme may be activated several fold by the protein activator which was discovered by Cheung((1971) J. Biol. Chem. 246, 2859-2869). The PII enzyme is refractory to this activator, and is not inhibited by the Ca2+ chelating agent, ethylene glycol bis (beta-aminoethyl ether)-N, N'-tetraacetate (EGTA). The activated activity of PI phosphodiesterase may be further stimulated by imidazole or NH+4, and inhibited by high concentrations of Mg2+. These reagents have no significant effect on either the PII enzyme or the basal activity of PI phosphodiesterase. Although both forms of phosphodiesterase can hydrolyze either cyclic AMP or cyclic GMP, they exhibit different relative affinities towards these two cyclic nucleotides. The PI enzyme appears to have much higher affinities toward cyclic GMP than cyclic AMP. Km values for cyclic AMP and cyclic GMP are respectively 1.7 and 0.33 mM for the non-activated PI phosphodiesterase; and 0.2 and 0.007 mM for the activated enzyme. Each cyclic nucleotide acts as a competitive inhibitor for the other with Ki values similar to the respective Km values. In contrast with PI phosphodiesterase, PII phosphodiesterase exhibits similar affinity toward cyclic AMP and cyclic GMP. The apparent Km values of cyclic AMP and cyclic GMP for the PII enzyme are approx. 0.05 and 0.03 mM, respectively. The kinetic plot with respect to cyclic GMP shows positive cooperativity. Each cyclic nucleotide acts as a non-competitive inhibitor for the other nucleotide. These kinetic properties of PI and PII phosphodiesterase of bovine heart are very similar to those of rat liver cyclic GMP and high Km cyclic AMP phosphodiesterases, respectively (Russel, Terasaki and Appleman, (1973) J. Biol. Chem. 248, 1334).     

Phosphatidylglycerol-modulated protein kinase activity from human spleen. II. Interaction with phospholipid vesicles

Phosphorylation of endogenous and artificial protein substrates by protein kinase P is stimulated by phosphatidylinositol or phosphatidylglycerol (D. J. Klemm, and L. Elias (1987) J. Biol. Chem. 262, 7580-7585; L. Elias and A. Davis (1985) J. Biol. Chem. 260, 7023-7028). Stimulation of protein kinase P activity required phospholipid vesicles rather than free phospholipid molecules. Protein kinase P activity increased as the phosphatidylinositol content of the vesicles was raised from 20 to 100%; no stimulation was detected below 20% phosphatidylinositol. This suggests that a vesicle surface rich in phosphatidylinositol is required for enzyme activation. Maximum activation of protein kinase P activity showed an optimum value with respect to phospholipid concentration, with both endogenous and artificial protein substrates. The phospholipid concentration at which optimal enzyme activity occurred shifted in response to the concentration of protein substrate, but not enzyme concentration. Therefore, the density of substrate molecules on the surface of phospholipid vesicles is a critical feature of protein kinase P stimulation. Binding of protein kinase P to vesicles was independent of micelle composition, but the binding of the artificial substrate, histone H2B, was specific for vesicles containing phosphatidylinositol or phosphatidylglycerol, and increased as the content of phosphatidylinositol was increased. Thus, an important feature of protein kinase P activation appeared to be the specific binding of protein substrate to phospholipid vesicles.     

Purification and partial characterisation of tentatively classified acid phosphatase from the earthworm Eisenia veneta

In order to use leakage of lysosomal acid phosphatase (AP) as a biomarker of stress to earthworms, more information about AP’s in earthworms are needed. This paper describes the details about tentatively classified APs in the earthworm Eisenia veneta. Two isoenzymes (enzyme I and II) of acid phosphatase (AP) and one alkaline phosphatase (enzyme III) from the earthworm E. veneta were separated by gel filtration. All three enzymes were further purified and concentrated on a Con A Sepharose 4B column. Enzyme I was inhibited by tartrate, showed an optimal pH range between 4.0 and 5.0 and was assumed to be of lysosomal origin. Enzyme II was the major enzyme showing the highest activity of the three enzymes. It was expected to be a lysosomal AP under physiological conditions. Enzyme II had a molecular mass 113 kDa and was composed of apparently identical polypeptide chains of 36 kDa each. This enzyme was inhibited by tartrate, showed an optimal pH in the range 6.0–7.5 and was slowly degraded at temperatures above 40°C. Enzyme III is not inhibited by tartrate and has a pH-optimum >9. The subcellular location under physiological conditions was assumed to be the cytosol.     

Mannitol-specific enzyme II of the bacterial phosphotransferase system. I. Properties of the purified permease

The integral membrane protein responsible for the transport and phosphorylation of D-mannitol in Escherichia coli, the mannitol-specific Enzyme II of the phosphotransferase system (Mr = 60,000), has been purified to apparent homogeneity using a modification of a previously published procedure (Jacobson, G. R., Lee, C. A., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 249-252). The purified enzyme was dependent on Lubrol PX and phospholipid for maximal activity. It catalyzed both the phosphoenolpyruvate- and the mannitol 1-phosphate-dependent phosphorylation of D-mannitol with high specificity for the accepting sugar and the phosphoryl donor. Both mannitol and mannitol 1-phosphate gave strong substrate inhibition at neutral pH in the transphosphorylation reaction catalyzed by the purified mannitol Enzyme II, while no substrate inhibition by mannitol was observed for the phosphoenolpyruvate-dependent reaction. The purified enzyme did not catalyze hydrolysis of mannitol 1-phosphate, a product of both reactions. Antibody directed against the mannitol Enzyme II inhibited the phosphoenolpyruvate-dependent activity to a greater extent than the transphosphorylation activity. Limited proteolysis with trypsin rapidly inactivated both purified and membrane-bound mannitol Enzyme II, and the purified protein was concomitantly cleaved into fragments with apparent molecular weights of about 29,000. These results show that although the mannitol Enzyme II is an integral membrane protein, a considerable portion of its polypeptide chain must also extend into a hydrophilic environment, presumably the cytoplasm.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in minimal deviation hepatoma 7288C. Immunological measurements in hepatoma tissue culture cells.

The mechanism of action of serum lipoproteins and 25-hydroxycholesterol on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells was investigated using antiserum against purified rat liver HMG-CoA reductase (Heller, R. A., and Shrewsbury, M. A. (1976)J. Biol. Chem. 251, 3815-3822). This antiserum cross-reacted with solubilized and membrane-bound HMG-CoA reductase from HTC cells. The enzymes from rat liver and HTC cells appeared antigenically identical. The increase in HMG-CoA reductase activity of HTC cells grown in medium which lacked serum lipoproteins was shown to be due to an increase in immunoprecipitable enzyme. In contrast, the 25-hydroxycholesterol suppression of reductase activity leads to a reduction in the antigenicity of the enzyme rather than a decrease in its number of molecules.     

Nonspecific lipid transfer protein in the assay of a membrane-bound enzyme CMP-N-acetyl-neuraminate:lactosylceramide sialyltransferase

CMP-N-acetylneuraminate:lactosylceramide alpha-2,3-sialyltransferase is tightly associated with the luminal side of the Golgi membrane as is its lipid substrate, lactosylceramide. In order to understand the kinetics, properties, and regulation of this enzyme, it is necessary to alter the amount and type of substrate in the membrane while minimizing changes in the membrane environment or in the conformation of the enzyme. Therefore, nonspecific lipid transfer protein, which accelerates the transfer of phospholipids, cholesterol, and glycosphingolipids between membranes was used to study the properties and kinetics of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase. These results are compared to those obtained in parallel experiments using detergent-solubilized substrate. Enzyme activity was increased four- to fivefold by transfer protein and was consistently higher than the activity measured in the presence of detergents. In contrast to the results obtained with detergents, the enzyme activity increased linearly with both Golgi protein and with incubation time for up to 60 min. The Km values for the water-soluble substrate, CMP-neuraminic acid, were virtually identical when determined in the presence of transfer protein (0.23 mM) or detergents (0.27 mM). On the other hand, the apparent Km values for the lipophilic substrate, lactosylceramide, were markedly different when determined in the presence of transfer protein (47.9 microM) or in the presence of detergents (1.2 microM). These observations suggest that transfer protein is a useful tool to study the properties and kinetics of membrane-bound enzymes when both the enzyme and substrate are components of the same membrane.     

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

A dipeptidyl peptidase (DPP) was purified to homogeneity using lys-ala-beta-naphthylamide, the standard substrate for DPP II. The enzyme is a monomer with a Mr of 70kDa, pl 5.2, and Km 5.0 microM. Its terminal amino acid sequence was XXLLYAIQKRLF and was not identical to that of any known protein. Although initially considered to be a DPP II, the enzyme differed in some properties from classical DPP IIs. It had a pH optimum of 7.9, was not active on X-pro-naphthylamides, the usual substrates of mammalian DPP II, but was active on arg-arg- and asp-arg-naphthylamides, substrates acted on by the DPP III class of enzymes. This enzyme therefore combines properties typical of both DPP II and III and differs from all previously described DPPs. Activity on lys-ala-beta-naphthylamide was most abundant during aggregation and its activity is consistent with processing specific peptides during development.     

The biosynthesis of highly branched N-glycans: studies on the sequential pathway and functional role of N-acetylglucosaminyltransferases I, II, III, IV, V and VI

At least 6 N-acetylglucosaminyltransferases (GlcNAc-T I, II, III, IV, V and VI) are involved in initiating the synthesis of the various branches found in complex asparagine-linked oligosaccharides (N-glycans), as indicated below: GlcNAc beta 1-6 GlcNAc-T V GlcNAc beta 1-4 GlcNAc-T VI GlcNAc beta 1-2Man alpha 1-6 GlcNAc-T II GlcNAc beta 1-4Man beta 1-4-R GlcNAc T III GlcNAc beta 1-4Man alpha 1-3 GlcNAc-T IV GlcNAc beta 1-2 GlcNAc-T I where R is GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAcAsn-X. HPLC was used to study the substrate specificities of these GlcNAc-T and the sequential pathways involved in the biosynthesis of highly branched N-glycans in hen oviduct (I. Brockhausen, J.P. Carver and H. Schachter (1988) Biochem. Cell Biol. 66, 1134-1151). The following sequential rules have been established: GlcNAc-T I must act before GlcNAc-T II, III and IV; GlcNAc-T II, IV and V cannot act after GlcNAc-T III, i.e., on bisected substrates; GlcNAc-T VI can act on both bisected and non-bisected substrates; both Glc-NAc-T I and II must act before GlcNAc-T V and VI; GlcNAc-T V cannot act after GlcNAc-T VI. GlcNAc-T V is the only enzyme among the 6 transferases cited above which can be essayed in the absence of Mn2+. In studies on the possible functional role of N-glycan branching, we have measured GlcNAc-T III in pre-neoplastic rat liver nodules (S. Narasimhan, H. Schachter and S. Rajalakshmi (1988) J. Biol. Chem. 263, 1273-1281). The nodules were initiated by administration of a single dose of carcinogen 1,2-dimethyl-hydrazine.2 HCl 18 h after partial hepatectomy and promoted by feeding a diet supplemented with 1% orotic acid for 32-40 weeks. The nodules had significant GlcNAc-T III activity (1.2-2.2 nmol/h/mg), whereas the surrounding liver, regenerating liver 24 h after partial hepatectomy and control liver from normal rats had negligible activity (0.02-0.03 nmol/h/mg). These results suggest that GlcNAc-T III is induced at the pre-neoplastic stage in liver carcinogenesis and are consistent with the reported presence of bisecting GlcNAc residues in N-glycans from rat and human hepatoma gamma-glutamyl transpeptidase and their absence in enzyme from normal liver of rats and humans (A. Kobata and K. Yamashita (1984) Pure Appl. Chem. 56, 821-832).