Characterization of the N-deacetylase domain from the heparan sulfate N-deacetylase/N-sulfotransferase 2

Heparin and heparan sulfate are linear sulfated polysaccharides that exert a multitude of biological functions. Heparan sulfate glucosaminyl N-deacetylase/N-sulfotransferase isoform 2 (NDST-2), a key enzyme in the biosynthesis of heparin, contains two distinct activities. This bifunctional enzyme removes the acetyl group from N-acetylated glucosamine (N-deacetylase activity) and transfers a sulfuryl group to the unsubstituted amino position (N-sulfotransferase activity). The N-sulfotransferase activity of NDST has been unambiguously localized to the C-terminal domain of NDST. Here, we report that the N-terminal domain of NDST-2 retains N-deacetylase activity. The N-terminal domain (A66-P604) of human NDST-2, designated as N-deacetylase (NDase), was cloned as a (His)(6)-fusion protein, and protein expression was carried out in Escherichia coli. Heparosan treated with NDase contains N-unsubstituted glucosamine and is highly susceptible to N-sulfation by N-sulfotransferase. Our results conclude that the N-terminal domain of NDST-2 contains functional N-deacetylase activity. This finding helps further elucidate the mechanism of action of heparan sulfate N-deacetylase/N-sulfotransferases and the biosynthesis of heparan sulfate in general.     

Distinct effects on heparan sulfate structure by different active site mutations in NDST-1

Heparan sulfate polymerization and modification take place in the Golgi compartment. The modification reactions are initiated by glucosaminyl N-deacetylase/N-sulfotransferase (NDST), a bifunctional enzyme that removes N-acetyl groups from selected N-acetyl-d-glucosamine units followed by N-sulfation of the generated free amino groups. Four isoforms of NDST have been identified. NDST-1 and -2 have a wide and largely overlapping tissue distribution, but it is not known if they can act on the same heparan sulfate chain. We have introduced point mutations into NDST-1 cDNA, which selectively destroy the N-deacetylase or N-sulfotransferase activity of the enzyme [Wei, Z., and Swiedler, S. J. (1999) J. Biol. Chem. 274, 1966-70 and Sueyoshi, T., et al. (1998) FEBS Lett. 433, 211-4]. Stable 293 cell lines expressing the NDST-1 mutants were then generated. Structural analyses of heparan sulfate synthesized by these cells and by cells overexpressing wild-type NDST-1 demonstrate that the N-deacetylation step is not only prerequisite but also rate-limiting, determining the degree of N-sulfation. Transfection of mutant NDST-1 lacking N-deacetylase activity had no effect on heparan sulfate sulfation, while cells expressing wild-type enzyme or NDST-1 lacking N-sulfotransferase activity both resulted in the production of oversulfated heparan sulfate. Since no increase in the amount of N-unsubstituted glucosamine residues was seen after transfection of the mutant lacking N-sulfotransferase activity, the results also suggest that two different enzyme molecules can act on the same glucosamine unit. In addition, we show that oversulfation of heparan sulfate produced by cells tranfected with wild-type NDST-1 or the mutant lacking N-sulfotranferase activity results in decreased sulfation of chondroitin sulfate.     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

Partial Purification and Characterization of Detergent-Solubilized N-Sulfotransferase Activity Associated with Calf Brain Microsomes

Calf brain 3'-phosphoadenosine 5'-phosphosulfate (PAPS):proteoheparan sulfate (PHS) N-sulfotransferase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3',5'-ADP-agarose as chromatographic supports. Sulfotransferase activity was followed by using 3'-phosphoadenosine 5'-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22- to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5-8. Apparent Km values for PAPS of 2.3 microM and 0.9 microM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and lysophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.     

Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF(164) to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF(164), reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin-ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin-ubiquitin tag from biotin-ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.     

Overexpression of different isoforms of glucosaminyl N-deacetylase/N-sulfotransferase results in distinct heparan sulfate N-sulfation patterns

Functional interactions of heparan sulfate (HS) with selected proteins depend on distinct saccharide sequences which are generated during biosynthesis of the polysaccharide. Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) catalyze both the N-deacetylation and N-sulfation reactions that initiate the modification of the (GlcNAc-GlcA)(n) polysaccharide backbone. The N-acetyl/N-sulfate exchange is restricted to certain regions of the polysaccharide chains, and only these can be further modified by glucuronyl C5-epimerization and O-sulfation at various positions. To investigate whether NDST isoforms influenced differently the structure of HS, murine NDST-1 was overexpressed in human kidney 293 cells, and the structure of the HS produced was compared to HS from NDST-2 overexpressing cells [Cheung, W. F., Eriksson, I., Kusche-Gullberg M., Lindahl, U., and Kjellén, L. (1996) Biochemistry 35, 5250-5256]. The level of N-sulfation increased from 40% in control cells to 60% and 80%, respectively, in NDST-1 and NDST-2 transfected cells. Interestingly, the increase in N-sulfation was accompanied by an increased chain length, while no effect on IdoA content or O-sulfation was seen. The most extended N-sulfated domains were found in HS synthesized by NDST-2 transfected cells. Since both the N-deacetylase and the N-sulfotransferase activities were lower in these cells than in the NDST-1 overexpressing cells, we conclude that, in addition to the level of enzyme expression, the NDST isoform also is important in determining the N-sulfation pattern in HS.     

Amine N-sulfotransferase

A highly purified amine N-sulfotransferase has been isolated from guinea pig liver that catalyzes sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate to one of a large number of either primary or secondary amines forming the appropriate sulfamate and adenosine 3',5'-bisphosphate. Amines as different as aniline, 2-naphthylamine, octylamine, 1,2,3,4-tetrahydroisoquinoline and 1,2,3,4-tetrahydroisoquinoline, desmethylimipramine, and cyclohexylamine serve as acceptors; the product of the last of these substrates is the sugar-substitute cyclamate. Amine N-sulfotransferase activity is dependent on the presence of an unprotonated amino group. The purified enzyme preparation also has O-sulfotransferase activities, suggesting that transfer to oxygen could represent an intrinsic function of the N-sulfotransferase.     

Peroxisomal beta-oxidation system of Candida tropicalis. Purification of a multifunctional protein possessing enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase activities

A multifunctional protein from oleate-grown cells of Candida tropicalis has been purified and partially characterized. A simple two-step purification has been developed involving ion-exchange chromatography followed by dye-ligand chromatography on blue Sepharose CL-6B. Homogeneous enzyme with a subunit Mr of 102 000 is obtained in 60% yield. The native relative molecular mass, determined by three different methods, yielded values which suggest that the enzyme is dimeric. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein revealed a single polypeptide band and reverse-phase high-performance liquid chromatography indicated a single component suggesting that this protein may consist either of two identical or very similar subunits. Three beta-oxidation activities, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase, co-purified with this protein. The ratio of the three beta-oxidation enzyme activities remained constant during purification and was unchanged by additional chromatographic methods (adsorption and affinity chromatography), thus indicating the multifunctional nature of this protein. Enzymatic staining of the purified protein for 3-hydroxyacyl-CoA dehydrogenase and epimerase, following electrophoresis in a polyacrylamide density gradient, further supported the multifunctionality of this protein. After isopycnic centrifugation of a particulate fraction from oleate-grown cells in a linear sucrose gradient the activities of all individual beta-oxidation enzymes cosedimented with catalase and with the glyoxylate bypass enzymes. This result demonstrated the peroxisomal localization of the multifunctional enzyme. The relationship of this multifunctional protein to the two bifunctional beta-oxidation enzymes isolated from peroxisomes of rat liver and from glyoxysomes of cucumber seeds is discussed.     

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

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

Nicotinamide adenine dinucleotide phosphate-malic enzyme of rat liver. Purification, properties, and immunochemical studies.

Rat liver malic enzyme (EC 1.1.1.40) was purified from livers of rats fasted and refed a high sucrose diet containing 1% desiccated thyroid powder. The purification was accomplished by a six-step procedure. The specific activity of the purified enzyme was increased 181-fold above that of the initial high speed supernatant of liver extracts. Slight additional purification of malic enzyme was achieved with preparative disc electrophoresis. The specific activities of the purified rat liver malic enzyme from the least two steps were between 28.0 and 30.5 units per mg of protein. Homogeneity of the purified enzyme was determined by disc and starch gel electrophoresis as well as sedimentation velocity and sedimentation equilibrium studies. The molecular weight and S20, w values of rat liver malic enzyme are 268,000 and 10.2, respectively. Amino acid analysis based on milligram of protein hydrolyzed yielded higher amounts of leucine and glutamic acid but lower quantities of alanine and voline per subunit than the corresponding Escherichia coli enzyme...     

Purification and properties of a lipolytic acyl-hydrolase from potato leaves.

A lipolytic acyl-hydrolase was purified 520-fold from an homogenate of potato leaves (Solanum tuberosum L. cv. Benimaru). The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.6 and a molecular weight of about 110,000. It had pH optima of 5.5 and 5.0, and Km values of 0.26 and 0.54 mM for monogalactosyldiacylglycerol and phosphatidylcholine, respectively. The pH dependences were altered by the addition of Triton X-100. No separation of these two hydrolyzing activities was achieved; the ratio of the specific activity of galactolipase to that of phospholipase (about 7/1) remained constant throughout the purification procedures. Both the activities were changed in parallel with each other by the addition of reagents and by heat treatment. The enzyme clearly catalyzed the deacylation of the several classes of galacto- and phospholipids. These results suggest that a single enzyme is responsible for both activities.     

Histone-lysine methyltransferase activity from sea-urchin embryo nuclei. Changes in substrate specificity upon purification

The S-adenosylmethionine:histone-lysine methyltransferase (EC 2.1.1.43) enzyme activity, present in the chromatin of sea-urchin embryo nuclei, has been purified about 300-fold with 30% overall yield. The initial activity in the nucleus transfers methyl groups to the epsilon-amino group of lysines and acceptor proteins are chromatin-bound H3 and H4 histones. In contrast, the purified enzyme activity transfers methyl groups to the arginines and acceptor proteins are soluble H3 and H4 histones. The two changes in substrate specificity do not occur at the same time. The variation of acceptor protein from chromatin-bound to soluble histones occurs at the first step, upon nuclei sonication, when no protein fractionation has yet been performed. At that step, lysine is still the only methylated side-chain. The variation of the methylated amino acid from lysine to arginine occurs gradually with increasing enzyme purification. The enzyme activity has a molecular mass of about 200 kDa. Saturation curves for H3 and H4 histones, used as substrate either individually or in total histones, and for AdoMet show no substantial dependence on enzyme purification. Maximal activity for the enzyme, at all purification levels, occurs at about pH 8 for all substrate histones. An increase in the relative concentrations of di- and trimethyllysine derivatives is observed with the more purified enzyme preparations, while the ratio of mono- and dimethylarginine derivatives remains constant. The data are taken as evidence that the same protein molecule is responsible for the two activities.     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Purification and properties of alpha-aminoadipate aminotransferase from rat kidney.

Previous studies with rat kidney preparations indicated that alpha-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are associated with a single protein. However, recent studies from our laboratory demonstrated that AadAT and KAT activities belong to two different proteins. AadAT from rat kidney supernatant fraction was purified by affinity chromatography to electrophoretic homogeneity. This rapid and efficient procedure improved the yield and the degree of purification over previously published methods and separated AadAT from KAT. The molecular weight of the enzyme was estimated to be 89,000 by Sephadex G-200 gel filtration chromatography. SDS-PAGE indicated that the enzyme is composed of two apparently identical subunits. Absorption spectra and the kinetic properties of AadAT are reported.     

金属螫合亲和层析纯化和复性重组人铜锌超氧化物歧化酶

将重组人铜锌超氧化物歧化酶（rhCu,Zn-SOD）包含体（经纯化后纯度达80％以上）以稀释法或透析法初步复性后,分别再经金属螯合亲和层析（MCAC）纯化。结果透析复性样品和稀释复性样品经MCAC纯化后的rhSOD比活是各自上柱前样品比活的2．2倍和5．3倍,蛋白回收率分别为64％和25％,同时两者活性回收率皆大于130％。表明目标蛋白rhSOD在经过MCAC纯化的同时又获得进一步复性。SDS-PAGE显示rhSOD为19kD的单一条带,纯度大于95％,比活达到5000u／mg左右,同时NBT生物活性染色鉴定显示出很强的超氧化物歧化酶活性。表明MCAC对于复性不完全的rhCu,Zn-SOD而言是一种纯化和使其进一步复性的简便省时且有效的方法。该方法为以包含体形式表达的基因重组蛋白的纯化和复性提供了新思路。     

Regulation of microsomal stearoyl-coenzyme A desaturase. Purification of a non-substrate-binding protein that stimulates activity.

Crude cytosolic fraction from rat liver was examined for proteins that may be involved in regulation of microsomal stearoyl-CoA desaturase activity. Gel filtration revealed the presence of several components that either stimulate or inhibit this enzyme. In addition, other components bind the acyl-CoA substrate, thus affecting observed activities in vitro. A protein that stimulates stearoyl-CoA desaturase but does not bind substrate was purified approx. 1100-fold. The purified protein had no visible absorption spectrum and an approximate mol.wt. of 26500. Maximal stimulation of desaturase activity occurred with less than 2 micrometer purified protein. The protein was heat-labile and exhibited neither catalase nor glutathione peroxidase activity. Addition of the cytosolic protein produced no effect on the desaturase reaction stoicheiometry; the proportions O2 consumed/NADH oxidized/stearoyl-CoA desaturated remained 1:1:1. Because the Km' for stearoyl-CoA was also unchanged by addition of the cytosolic protein, no change in substrate affinity was suggested. Furthermore addition of the cytosolic protein also produced no effect on desaturase inhibition by oleoyl-CoA, which suggested that the protein does not simply relieve apparent product inhibition. These results indicate that, in analogy to other cytosolic proteins that stimulate microsomal oxidase activities, the protein may have a regulatory function, perhaps related to activity modulation via organization of the multienzymic desaturase in the membrane.     

Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.