Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Studies on the "aerobic" acetyl-coenzyme A synthetase of Saccharomyces cerevisiae: purification, crystallization, and physical properties of the enzyme.

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis.Sedimentation velocity runs revealed a single symmetric peak with an s_20,w value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 ± 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 ± 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic S. cerevisiae is composed of three subunits of identical or nearly identical size.     

Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Purification and characterization of the two molecular forms of membrane acid protease from Aspergillus oryzae.

Two forms (M1 and M2) of the membrane-bound acid protease of Aspergillus oryzae have been purified by extraction with Triton X-100, washing with cold acetone, and repeated gel filtration on Bio-Gel A-15 m in the presence and absence of Triton X-100. The purified membrane enzymes, M1 and M2, moved as a single band in acrylamide gel electrophoresis and had apparent molecular weights of 150 000 and 60 000, respectively, as estimated by sodium dodecyl sulfate/acrylamide gel electrophoresis. These two membrane enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They immunologically cross-reacted with each other and with an extracellular acid protease from A. oryzae, and contained carbohydrate, ranging from 52.5 to 80.5% and comprising three hexoses, glucose, galactose, and mannose. While these catalytic, chemical and immunological properties are similar to those of the extracellular acid protease from A. oryzae, both membrane enzyme differed in their hydrophobic properties from external enzymes. Thus they are activated by the detergent Triton X-100 and some polar lipids.     

Purification and properties of galactokinase from Saccharomyces cerevisiae.

Galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) was purified to homogeneity with a 50% yield from cells of Saccharomyces cerevisiae which were fully induced for the production of the galactose metabolizing enzymes. The purification was accomplished by:(a) ammonium sulfate fractionation, (b) streptomycin sulfate precipitation. (c) DEAE-cellulose chromatography, (d) hydroxylapatite chromatography, and finally (e) Bio-Gel A-0.5 m gel filtration. The resulting preparation of galactokinase was judged to be at least 95% pure by the following criteria: (a) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) ultracentrifuge analysis, (c) nondissociating polyacrylamide gel electrophoresis, and (d) Bio-Gel A-0.5 m gel filtration. The purified enzyme preparation was used to determine the Km values for the two substrates, galactose and ATP, which were found to be 0.60 and 0.15 mM, respectively. Vmax was also determined and found to be 3.35 mmol/h/mg. This corresponds to a turnover rate of 3350 molecules of galactose phosphorylated/min/enzyme molecule. The effect of pH on the galactokinase-catalyzed phosphorylation of galactose was determined; the results showed the pH optimum of the reaction to be in the range of pH 8.0 to 9.0. The enzyme is highly specific for galactose since galactokinase did not appear to phosphorylate any of the other sugars tested at a rate greater than 0.5% of the rate of galactose phosphorylation. Amino acid analysis was performed on the enzyme preparation and the results were used to calculate the partial specific volume (v) of 0.736. The NH2-terminal sequence was determined for the first 3 residues. The molecular weight and subunit composition were determined by ultracentrifugation and polyacrylamide gel electrophoresis under dissociating and nondissociating conditions. The data obtained indicated that galactokinase is a monomeric protein of molecular weight 58,000.     

Bovine renal glomerular basement membrane. Isolation and characterization of a glycoprotein component.

Bovine glomerular basement membrane was extracted with 6 M guanidinium chloride and the soluble material fractionated on a Bio-Gel A-1.5m column in 1% Na dodecyl-SO4. A single component was obtained by reduction of a selected column fraction with 2-mercaptoethanol followed by chromatography on an analytical Bio-Gel A-1.5m column and shown to be homogenous by electrophoresis and ultracentrifugation. It consists of 90% protein and 8.6% carbohydrate by weight. The amino acid composition is characterized by the presence of low amounts of hydroxyproline and hydroxylysine, and substantial amounts of aspartic acid, glutamic acid, half-cystine, and glycine. It contains all the monosaccharide constituents present in the whole basement membrane indicating the presence of both heteropolysaccharide and disaccharide units; the presence of the latter unit was demonstrated unequivocally by ion exchange chromatography. The component contains 1 heteropolysaccharide unit and 4 dissaccharide units/molecule of Mr equals 70,000. The molecular weight of component VII was determined by several methods. Molecular weight values of 68,000 +/- 3,000 and 72,000 +/- 2,000 were determined in 6 M guanidinium chloride by the methods of sedimentation equilibrium and gel filtration chromatography, respectively, and values of 136,000 +/- 3,100 and 140,000 +/- 2,000 were determined in 1% Na dodecyl-SO4 by the methods of polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. Circular dichroism spectra indicate that component VII assumes a random coil conformation in 6 M guanidinium chloride and a more disordered conformation in 1% Na dodecyl-SO4 than standard proteins used in calibration of polyacrylamide gels and gel filtration column. These results indicate that the minimal molecular weight of component VII is about 70,000 and that the anomalous behavior in Na dodecyl-SO4 is due in part to its conformation.     

Purification, preliminary characterization, and immunological comparison of hog lens leucine aminopeptidase (EC 3.4.11.1) with hog kidney and beef lens aminopeptidases

Leucine aminopeptidase (LAP) was purified from hog lenses by application of the Himmelhoch procedure for isolation of hog kidney LAP [S. R. Himmelhoch (1970) in Methods in Enzymology (Perlmann, G. E., and Lorand, L., eds.), Vol. 19, pp. 508-513, Academic Press, New York.] This involved treating crude hog lens homogenates with hexadecyltrimethylammonium bromide, DEAE-cellulose adsorption and elution, ammonium sulfate fractionation (53-84% of saturation), and gel filtration on a Bio-Gel A-1.5m column. Purifications ranging from 2080- to 4700-fold with activity yields from 28 to 100% were achieved. The hog lens LAP appeared homogeneous by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Bio-Gel chromatography of the native enzyme and SDS-PAGE of dimethylsuberimidate-crosslinked LAP indicated a molecular weight of 326,000. SDS-PAGE of untreated LAP showed a subunit weight of 54,000, consistent with a hexameric enzyme structure. By immunodiffusion, LAP from hog lens and kidney were identical while hog lens and beef lens enzymes demonstrated only partial identity. Electrophoresis of the native enzymes showed a slightly lower mobility for the hog lens LAP than for beef LAP at pH 8.7.     

Purification and initial characterization of the proteasome from the higher plant Spinacia oleracea.

The proteasome (multicatalytic protease complex), a high molecular weight protein complex, has been purified from spinach leaves by successive chromatography on DEAE-cellulose, Bio-Gel A-1.5m, DEAE-TOYOPEARL 650C, and DEAE-5PW. The molecular mass was estimated to be 850 kDa by gel filtration. Polyacrylamide gel electrophoresis of the proteasome gave a single protein band under nondenaturing conditions and at least 10 bands in the range of 21-32 kDa in the presence of sodium dodecyl sulfate. By electron microscopy after negative staining with uranyl acetate, the proteasome from spinach appeared as symmetrical ring-shaped particles. The substrate specificity of proteasomes indicates that they contain at least three types of activity, namely, chymotrypsin-like, Staphylococcus aureus V8 protease-like, and trypsin-like activities. The former two activities were enhanced by poly-L-lysine or sodium dodecyl sulfate. Moreover, we examined the immunological reactivities of proteasomes from various eukaryotes. As a result, cross-immunoreactivities of some subunits were observed. These properties of the proteasome are similar to those of proteasomes isolated from various other eukaryotic sources.     

Purification and characterization of a murine basement membrane collagen-degrading enzyme secreted by metastatic tumor cells

A type IV collagen-degrading enzyme activity secreted by a highly metastatic mouse tumor was purified by concanavalin A- and type IV collagen-agarose affinity chromatographies followed by gel filtration on Bio-Gel A-0.5 m. The apparent molecular weight of the enzyme was 160,000 but about 70,000 when Triton X-100 was added to the column buffer. The purified enzyme protein was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptide chains of about 68,000 and 62,000 daltons. The enzyme activity could be increased by preincubation with trypsin and it is possible that the two chains represent latent and active enzyme forms. The enzyme activity was not reduced in the presence of dithiothreitol, it had a pH optimum of 7.6 and was inhibited by EDTA but not N-ethylmaleimide, phenylmethylsulfonyl fluoride, or Trasylol. The inhibition with EDTA was reversible. The pro-alpha 1(IV) and pro-alpha 2(IV) chains of the type IV procollagen substrate were both degraded at a similar rate to form two pairs of degradation fragments corresponding in molecular weights to about 70 and 30% of the original size chains. The presence of Triton X-100 increased slightly the activity of the enzyme and diminished the reduction of its activity upon freezing, indicating that the enzyme is a hydrophobic protein.     

Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

R plasmid dihydrofolate reductase with a dimeric subunit structure

Dihydrofolate reductase specified by plasmid R483 from a trimethoprim-resistant strain of Escherichia coli has been purified 2,000-fold to homogeneity using dye-ligand chromatography, gel filtration, and polyacrylamide gel electrophoresis. The protein migrated as a single band on nondenaturing polyacrylamide gel electrophoresis and had a specific activity of 250 mumol/mg min(-1). The molecular weight was estimated to be 32,000 by gel filtration and 39,000 by Ferguson analysis of polyacrylamide gel electrophoresis. When subjected to electrophoresis in the presence of sodium dodecyl sulfate, the protein migrated as a single 19,000-molecular weight species, a fact that suggests that the native enzyme is a dimer of similar or identical subunits. Antibody specific for R483-encoded dihydrofolate reductase did not cross-react with dihydrofolate reductase encoded by plasmid R67, T4 phage, E. coli RT500, or mouse L1210 leukemia cells. The amino acid sequence of the first 34 NH2-terminal residues suggests that the R483 plasmid dihydrofolate reductase is more closely related to the chromosomal dihydrofolate reductase than is the enzyme coded by plasmid R67.     

A novel phosphodiesterase from cultured tobacco cells.

A novel phosphodiesterase was purified from cultured tobacco cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5'-phosphate, p-nitrophenyl thymidine 3'-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides, and poly(adenosine diphosphate ribose), which is a polymer synthesized from NAD+. However, it did not hydrolyze highly polymerized polynucleotides. The molecular weight of the native enzyme was estimated as 270 000 to 280 000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with molecular weights calculated to be 75 000. The enzyme did not require divalent cations for activity being fully active in the presence of ethylenediaminetetraacetic acid. The pH optimum for the enzyme was approximately 6 with p-ni-trophenyl thymidine 5'-phosphate or adenosine cyclic 3',5'monophosphate, and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5'-phosphate gave two apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least two active sites.     

Mycoplasma Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System: Purification and Characterization of Enzyme I

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble phosphocarrier protein (HPr), and a soluble enzyme I. The soluble enzyme I was purified by ammonium sulfate fractionation; Bio-Gel P-10 gel filtration; acid precipitation; diethylaminoethyl-Bio-Gel A; and Bio-Gel HTP column chromatography. The enzyme I was shown to be homogeneous by electrophoresis in a pH 8.9 non-sodium dodecyl sulfate gel and by isoelectric focusing. Whereas the protein moved as a single component in both the non-sodium dodecyl sulfate gel and isoelectric focusing, on sodium dodecyl sulfate gels, it moved as three subcomponents. The molecular weights of the three subunits, alpha, beta, and gamma, were 44,500, 62,000 and 64,500, respectively. The holoprotein moved as a single component, in the region of 220,000 daltons, in a Bio-Gel A 0.5-agarose column. The molar ratio of subunits was estimated to be 2alpha:1beta:1gamma. The elution characteristics on a diethylaminoethyl column at pH 7.4 and 6.8, acid precipitation data, and amino acid composition indicated that the protein is acidic. Isoelectric focusing occurred at pH 4.8. N-terminal amino acids determined by the dansyl chloride method indicated that glycine, alanine, and tyrosine are N-terminal amino acids of the three subunits. Although the protein was stable for at least 14 months at -20 degrees C, it was irreversibly inactivated by the thiol reagent N-ethyl-maleimide.     

Isolation and characterization of ceramide glycanase from the leech, Macrobdella decora

We have devised a simple method for achieving 890-fold purification of ceramide glycanase with 17% recovery from a North American leech, Macrobdella decora. The method includes water extraction, ammonium sulfate fractionation, and chromatography on octyl-Sepharose, Matrex gel blue A, and Bio-Gel A-0.5m columns. The final preparation showed one major protein band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using Bio-Gel A-0.5m filtration, the native enzyme was found to have a molecular mass of 330 kDa. With GM1 as substrate, the optimum pH of this enzyme was determined to be 5.0; the enzyme was stable between pH 4.5 and 8.5. Zn2+ at 5 mM and Cu2+, Ag+, and Hg2+ at 1 mM strongly inhibited the hydrolysis of GM1 by ceramide glycanase. The ceramide glycanase released the intact glycan chain from various glycosphingolipids in which the glycan chain is linked to the ceramide through a beta-glucosyl linkage. This enzyme also cleaved lyso-glycosphingolipids such as lyso-GM1 and lyso-LacCer and synthetic alkyl beta-lactosides. Among seven alkyl beta-lactosides tested, the enzyme only hydrolyzed the ones with an alkyl chain length of four or more carbons. The enzyme also hydrolyzed 2-(octadecylthio)ethyl O-beta-lactoside and 2-(2-carbomethoxyethylthio)ethyl O-beta-lactoside. p-Nitrophenyl, benzyl, and phytyl beta-lactosides, on the other hand, were not hydrolyzed. These results suggest that the enzyme can recognize the hydrophobic portion of glycolipid substrates. The fact that 2-(2-carbomethoxyethylthio)ethyl O-beta-N-acetyllactosaminide and DiGalCer were refractory to the enzyme indicated that in the substrate the first sugar attached to the hydrophobic chain cannot be N-acetylglucosamine and galactose. Furthermore, dodecyl maltoside, Gal alpha 1----6Glc beta Cer, and the LacCer in which the --CH2OH of the galactose was converted into --CHO were also resistant to the enzyme, and Man beta 1----4 Glc beta Cer was hydrolyzed at a much slower rate than LacCer. These results indicate that the nature and the linkage of the sugar attached to the glucose have a profound effect on the action of this enzyme. The hydrolysis of glycosphingolipids by ceramide glycanase is stimulated by bile salts. Among various bile salts tested, sodium cholate at a concentration of 1 microgram/microliter was found to be most effective in stimulating the hydrolysis of various glycosphingolipids with the exception of LacCer. For LacCer, sodium taurodeoxycholate at a concentration of 2-3 micrograms/microliters was most effective. Tween 20, Nonidet P-40, and Triton X-100 did not stimulate the hydrolysis of GM1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa

An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose. The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4. It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+. Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide. The enzyme does not attack RNA and is inhibited by it. Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000. The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation. The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis. The enzyme degrades denatured DNA about 20 times faster than native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Purification and properties of beta-N-acetylglucosaminidase from Escherichia coli.

beta-N-acetylglucosaminidase (EC 3.2.1.30) has been purified from Escherichia coli K-12 to near homogeneity based on polyacrylamide gel electrophoresis in both 0.5% sodium dodecyl sulfate and in 6 M urea at pH 8.5. The purified enzyme shows a pH optimum of 7.7 and the Km for p-nitrophenyl-beta-D-2-acetamido-2-deoxyglucopyranoside is 0.43 mM. The molecular weight of this enzyme, determined by both Sephadex gel filtration and by sodium dodecyl sulfate gel electrophoresis, is equivalent to 36,000. It is shown to be a soluble cytoplasmic enzyme. Studies on the substrate specificites of the purified enzyme indicate that this enzyme is an exo-beta-N-acetylglucosaminidase.     

delta-Aminolevulinic acid synthetase from rat liver mitochondria. Purification and properties.

delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.