Purification and characterization of alkaline phosphatase in cultured rat liver cells.

Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.     

Purification and characterization of alkaline phosphatase from cultured rat ascites hepatoma cells.

Alkaline phosphatase of cultured rat ascites hepatoma cells has been purified by butanol extraction, DEAE-cellulose column chromatography, gel filtration through Sephadex G-200, concanavalin A-Sepharose affinity chromatography, and polyacrylamide gel electrophoresis. Affinity chromatography confirmed the glycoprotein nature of alkaline phosphatase from cultured rat ascites hepatoma cells. Electrophoresis on polyacrylamide gels of various concentrations indicated a molecular weight of 290,000. The molecular weight of the subunit was estimated to be 72,000 by SDS-polyacrylamide gel electrophoresis. These findings suggest that alkaline phosphatase of cultured rat ascites hepatoma cells is a tetramer with a subunit molecular weight of 72,000.     

Inhibitor of pyrimidine metabolism from tumor tissues

Inhibitors of normal rat liver 5′-nucleotidase and dUMP kinase $\text{in vitro}$ were found in rapidly proliferating tissues, such as Yoshida sarcoma. Two inhibitors were separated from Yoshida sarcoma by zone electrophoresis, gel filtration on Sephadex G-200 and DEAE-cellulose column chromatography. One inhibited both 5′-nucleotidase and dUMP kinase, while the other inhibited only dUMP kinase. These inhibitors were not detectable in normal rat liver. They were induced in regenerating rat liver and present in rapidly proliferating tissues, such as Yoshida sarcoma and Ehrlich ascites tumor and rat marrow cells. These inhibitors were heat labile. One had a large molecular weight (500,000>) and the other a small molecular weight ($\text{Ca}\text{.}$ 50,000).     

Purification of the phosphofructokinase regulatory factors

In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.     

Isolation and characterization of a sulfated glycoprotein from rabbit uterus.

Crude glycoproteins were extracted with 0.15 M NaCl from the pooled endometrial scrapings of rabbit uteri after treatment with estrogen. The crude glycoproteins were fractionated with ammonium sulfate, followed by DEAE-cellulose column chromatography, treatment with CM-Sephadex C-25 and gel filtration on Sephadex G-200. Subsequently, purification of an acidic glycoprotein was carried out by gel filtration on Sephadex G-200 and then Sepharose 4B. The results of electrophoresis and enzymatic digestion, together with analytical data and the infrared spectrum indicated that the acidic glycoprotein was a sulfated glycoprotein.     

The aconitase of yeast. II. Crystallization and general properties of yeast aconitase.

Yeast aconitase [citrate (isocitrate) hydro-lyase, ED 4.2.1.3], inductively formed by Candida iipolytica in the presence of fluoroacetate, was purified approximately 100-fold by Sephadex G-100 gel filtration and DEAE-Sephadex column chromatography, yielding dark-brown needle crystals. The crystalline aconitase was homogenious as judged by polyacrylamide gel electrophoresis and sedimentation by ultracentrifugation. The enzyme showed maximal activity at pH 8.0 and at 55 degrees. It has an S20, W of 5.03 S, a molecular weight of 68,500 and an isolectric point of pH 4.2. The presence of 2.10 moles of iron per mole of the enzyme was demonstrated by atomic absorption spectroscopy.     

Purification of the nitrogenase proteins from Clostridium pasteurianum

A method is described for the purification of the nitrogenase proteins from Clostridium pasteurianum by two polyethylene glycol precipitations and chromatography on columns of DEAE-cellulose, Sephadex G-100 and Sephadex G-200. The Mo-Fe protein and the Fe protein have been purified 70–80-fold from the crude extract, and they appear essentially pure when tested by anaerobic polyacrylamide gel electrophoresis.     

Purification of the prothoracicotropic hormone from the tobacco hornworm Manducasexta

Standard biochemical procedures were used to purify the prothoracicotropic hormone (PTTH) 4400 fold from whole head extracts of $\text{Mandura}$$\text{sexta}$ fifth instar larvae. Hormonal activity was bioassayed by injection into neck-ligated fourth instar larvae. The hormone was stable to heating at 85°C. Ammonium sulfate and acetone fractionation provided a crude preparation which showed dose-dependent activity in the bioassay. Chromatography on Sephadex G-100, DEAE-Sephadex, and hydroxylapatite gave a preparation with 2.6 $\text{Manduca}$ PTTH units/μg protein (4400-fold purification). Activity was sensitive to proteolytic enzymes. Further purification by preparative electrophoresis gave a preparation which migrated as a single band in two polyacrylamide gel electrophoresis systems. A molecular weight estimate of 25,000 Daltons was obtained for this bands on SDS polyacrylamide gels.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Galactosyltransferase: confirmation of equilibrium-ordered mechanism.

A thermostable protein that strongly inhibits the soluble $\text{E. coli}$ D-alanine carboxypeptidase was isolated from a cell-free extract of $\text{E. coli B}$. The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg⁺⁺ or Na⁺ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of $\text{E. coli}$ or the growth of the bacteria.     

Brain pyridoxal kinase. Purification and characterization

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.     

Identification and purification of a soluble adenosine triphosphatase from Tetrahymena pyriformis .

An unusual ATPase isolated from the postribosomal supernatant fraction of $\text{Tetrahymena pyriformis}$ has been purified to homogeneity. The purification procedure consisted of protamine sulfate and heat treatment; column chromatography successively on phosphocellulose, DEAE-cellulose and Sephadex G-150; and isoelectric focusing. The pure enzyme has a molecular weight of 89,000 and requires either Ca²⁺ or Ba²⁺ for maximum activation. Nucleoside triphosphates are hydrolized at decreasing rates in the order: ATP > GTP > ITP > CTP > UTP. The K_m for ATP is 2.5 mM. Because of its properties the enzyme is tentatively classified as a soluble Ca²⁺-activated ATPase.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Properties of two molecular forms of beta-glucuronidase from the mollusc Littorina littorea L.

The occurrence of the two molecular forms, I and II, in the beta-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and II of beta-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4--4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p-nitrophenyl beta-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein beta-D-glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This beta-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with beta 1 leads to 3 linkages; it had practially no activity on certain glycosaminoglycans. Hg2+ and glucaro-1,4-lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.     

Purification and properties of oxytocinase (cystine amino-peptidase) from monkey placenta.

Oxytocinase (cystyl-aminopeptidase) [EC 3.4.11.3] was isolated from monkey placenta in a purified form by a six-step prodedure comprising extraction from monkey placenta homogenate, ammonium sulfate fractionation, repeated chromatography on hydroxylapatite, chromatography on a column of DEAE-cellulose and gel filtration on a column of Sephadex G-200. The purified enzyme showed a single band on polyacrylamide disc electrophoresis. Oxytocin was inactivated by this enzyme preparation. The enzyme hydrolyzed several aminoacyl-beta-naphthylamides. A terminal amino group was required for enzyme activity. The molecular weight of the purified enzyme was estimated to be 87,000 by gel filtration and 83,000 by sodium dodecyl sulfate gel electrophoresis. Other properties of the enzyme, the effects of metal ions and various chemical reagents on the enzyme activity, the pH optimum, and Km values for a number of aminoacyl-beta-naphthylamides were also examined.     

Purification and characterization of the tween-hydrolyzing esterase of Mycobacterium smegmatis.

An esterase hydrolyzing Tween 80 (polyoxyethylene sorbitan monooleate) was purified from sonicated cell lysates of Mycobacterium smegmatis ATCC 14468 by DEAE-cellulose, Sephadex G-150, phenyl Sepharose, and diethyl-(2-hydroxypropyl) aminoethyl column chromatography and by subsequent preparative polyacrylamide gel electrophoresis. The molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 41,000 by gel filtration on a Sephadex G-150 column. The esterase contained a single polypeptide. The esterase was stable to heat treatment at 100 degrees C and to a wide range of pH. The temperature and pH optima for the hydrolysis of Tween 80 were 50 degrees C and 8.3, respectively. The esterase had a narrow substrate specificity; it exhibited a high activity only on compounds having both polyoxyethylene and fatty acyl moieties, such as Tweens. Monoacylglyceride was hydrolyzed more slowly by this esterase and this enzyme exhibited a nonspecific esterase activity on p-nitrophenyl acyl esters, especially those having short chain acyl moieties. The Km and Vmax were 19.2 mM and 1,670 mumol/min per mg of protein for Tween 20, 6.6 mM and 278 mumol/min per mg of protein for Tween 80, and 0.25 mM and 196 mumol/min per mg of protein for p-nitrophenyl acetate, respectively. Observations of the effects of various chemical modifications on the activity of the esterase indicated that tyrosine, histidine, arginine, and methionine (with tryptophan) residues may be active amino acids which play important roles in the expression of Tween 80-hydrolyzing activity of the enzyme.     

Isolation of an inhibitor of ß-glucuronidase from porcine sublingual gland

An inhibitor of ß-glucuronidase was isolated from porcine sublingual gland by successive fractionation of trypsin extracts of the latter on Sephadex G-100, DEAE-cellulose, Sephadex G-200, and DEAE-cellulose. Its purity and homogeneity were established by DEAE-cellulose column chromatography, ultracentrifugation, and electrophoresis on cellulose-acetate membrane. The sedimentation coefficient of the purified ß-glucuronidase inhibitor was 3.75 S (S₂₀⁰, w), and the molecular weight was determined to be 340 000 from Sephadex G-200 column chromatography. The inhibitor contained 17.5% protein, 20.8% total hexoses, 19.9% hexosamine, 21.8% N-acetylneuraminic acid, and 9.6% fucose. The inhibition was non-competitive, and it was completely suppressed by the addition of NaCl, KCl, Na₂SO₄, or CaCl₂, respectively.     

Studies on two isozymes of aconitase from Bacillus cereus T. III. Enzymatic properties.

The present communication describes a comparative study of some enzymatic properties of an early and a late aconitase (EC.4.2.1.3) present in $\text{Bacillus}\text{cereus}$ T cells of 5 and 12 hr culture age, respectively. The activity of both enzymes increased linearly with increase in enzyme concentration. They demonstrated similar pH *7.5) and temperature (30 C) optima, but differed in their activation energy and affinity for substrate. Late aconitase had higher activation energy (16,100 cal) as compared to early aconitase (9,200 cal). Early aconitase showed a Km value of 100 × 10^?4M for sodium citrate and 33.3 × 10^?4M for isocitrate. Late aconitase exhibited 5 to 7 times greater affinity for citrate and isocitrate yielding Km values 14 × 10^?4M and 7 × 10^?4M, respectively. On the basis of available evidence, it is suggested that early and late aconitase present in 5 and 12 hr aged cells of $\text{Bacillus}\text{cereus}$ T behave as isozymes, and may be designated as aconitase (EC.4.2.1.3) isozyme I and aconitase (EC.4.2.1.3) isozyme II, respectively. The significance of their plausible role during growth and sporulation has been discussed.     

Extraction of pure ribosomal protein and removal of Coomassie blue from acrylamide gels

A method has been developed to recover pure ribosomal proteins from two-dimensional polyacrylamide electrophoresis slabs. Proteins and Coomassie blue were extracted from the gels and separated by passing them through a Sephadex G-25 column. All the steps were performed in the presence of 66% ($\text{v}\text{v}$) acetic acid. The technique has been applied to Escherichia coll ribosomal proteins labeled with ¹²⁵I.     

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300 000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and K_m value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60°C for 10 min.