The characterization of multiple forms of kynurenine formidase in Drosophila melanogaster.

Two enzymic forms of kynurenine formamidase (EC 3.5.1.9) from Drosophila melanogaster were separated and partially purified by pH fractionation, (NH4) 2SO4 fractionation and Sephadex G-75 gel filtration. The enzymes were also separated by DEAE-cellulose ion-exchange chromatography and distinguished by their different rates of thermal inactivation. The multiple forms are termed formamidase I and formamidase II. The molecular weight of formamidase I as measured by Sephadex G-75 chromatography is 60 000 and that of formamidase II is 31 000. The pH optima are broad, ranging between 6.7 and 7.8 for formamidase I and 6.5 and 8.0 for formamidase II. The apparent Km values are 5-10(-3) and 0.83-10(-3) M, resepctively. The possibility that formamidase II is an active subunit of formamidase I is discussed, although neither enzyme will convert to the other when separated and rechromatographed. Eight organisms were tested for the presence or absence of multiple forms of formamidase. Drosophila melanogaster and Drosophila virilis have both enzymes; cow, chicken, yeast and housefly have formamidase I only, and mouse and frog have formamidase II only.     

Studies on the cholinesterase forms present in the ascitic fluid of Ehrlich tumour.

Cholinesterase activity was detected in the ascitic fluid of Ehrlich tumour and studied in a comparative manner in relation to that found in mice plasma. Enzymes from both sources were characterized with respect to optimum pH, substrate concentration and quinidine inhibition. After gel filtration by Sephadex G-200 and Sepharose 6B, two enzyme forms were observed in ascitic fluid as well as in mice plasma: a large form (L) and a small form (S) presenting molecular weights of 191 000, and 224 000 daltons for L forms and 71 000 and 69 000 daltons for S forms respectively. Concanavalin A interacts with both molecular forms, suggesting a glycoprotein nature for these enzymes.     

Characterization of two acid proteinases found in rabbit skin homografts.

Two types of acid proteinase activity found in rabbit skin homografts were characterized by studying the effect of temperature, pH and polyacrylamide-gel electrophoresis. Their chromatographic behaviour was characterized on DEAE-cellulose, Sephadex G-75, G-100 and G-200, and their molecular weights were estimated by gel filtration. One of the acid proteinases in the homograft resembled cathepsin D (EC 3.4.23.5) of normal skin. The other acid proteinase differed from cathepsin D with respect to heat inactivation, pH optimum and molecular weight; it was not inactivated on heating at 60 degrees C for 60 min, its pH optimum was 2.5 and its molecular weight measured by Sephadex G-100 chromatography was 100 000. In all these respects, the heat-stable proteinase resembles cathepsin E (EC 3.4.23.5) of rabbit polymorphonuclear leucocytes.     

Characterization of the multiple forms and main component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Multiple forms of dextransucrase (sucrose:1.6-alpha-D-glucan 6-alpha-D-glucosyltransferae EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F strain were shown by gel filtraton and electrophoretic analyses. Two components of enzyme, having different affinities for dextran gel, were separated by a column of Sephadex G-100. The major component voided from the Sephadex column was treated with dextranase and purified to an electrophoretically homogeneous state. The ]urified enzyme had a molecular weight of 64 000-65 000, pI value of 4.1, and 17% of carbohydrate in a molecule. EDTA showed a characteristic inhibition on the enzyme while stimulative effects were observed by the addition of exogenous dextran to the incubation mixture. The enzyme activity was stimulated by various dextrans and its Km value was decreased with increasing concentration of dextran. The purified enzyme showed no affinity for a Sephadex G-100 gel, and readily aggregated after the preservation at 4 degrees C in a concentrated solution.     

Possibility of shape conformers of the protein inhibitor of the cyclic adenosine monophosphate dependent protein kinase.

The heat-stable, protein inhibitor of the cyclic adenosine monophosphate (cAMP) dependent protein kinase [Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E., & Krebs, E (1971a) J. Biol. Chem. 246, 1977-1985] has been purified to homogeneity from rabbit skeletal muscle by preparative electrophoresis. Employing a more sensitive assay system, we detected multiple charged forms of the inhibitor on diethylaminoethyl chromatography; the form that has been further characterized is the predominant species in skeletal muscle comprising greater than 70% of the total. The apparent molecular weight of the protein inhibitor, as determined by Sephadex G-75 gel exclusion chromatography, is 22 000 in initial cellular extracts and at all stages during the purification prior to the final purification step of preparative gel electrophoresis, after which the homogeneous protein exhibits a molecular weight of 11 000. These two forms are designated I and I', respectively. The I form migrates with an apparent molecular weight of 10 000 on nondenaturing gel electrophoresis and of 10 500-11 500 on sodium dodecyl sulfate (NaDodSO4) gel electrophoresis; the I' form migrates with an apparent molecular weight of 6500-8300 on NaDodSO4 electrophoresis and has a minimum molecular weight of 10 400 by amino acid analysis. Taking into account the anomalous behavior displayed by low molecular weight proteins with the various techniques employed, we suggest that the I and I' forms of the protein inhibitor may represent shape conformers.     

Zinc binding in cow''s milk and human milk

In both cow's milk and human milk, zinc was associated with proteins of high molecular weight (greater than 100 000), as judged by analysis with Sephadex G-75. Precipitation of the casein at pH 4.6 and filtration of the resultant acid whey on Sephadex G-25 led, however, to the recovery of about 90% of the zinc as a compound of low molecular weight, which was tentatively identified as zinc citrate. Over 95% of the zinc of cow's milk was sedimented with the casein micelles on ultracentrifugation. Filtration of these micellar caseins on Sephadex G-150 gave two peaks containing zinc, which corresponded to aggregates of alpha-casein-kappa-casein and of alpha-casein-beta-casein. Ultracentrifugation of human milk sedimented only approx. 40% of total zinc. Analysis of sediment and supernatant on Sephadex G-150, however, indicated that about 85% of the zinc was associated with a protein complex of molecular weight greater than 150 000. The major protein of this complex was identified as lactoferrin. A minor zinc-binding component of average molecular weight 30 000 was also observed in the supernatant. The results indicated that zinc is bound to different macromolecules in cow's and human milk. This may be a factor affecting the bioavailability to the human infant of zinc from the two milks, and it is suggested that in human milk lactoferrin may be involved in the uptake of zinc.     

Protein kinases from liver mitochondria of tumour-bearing rats.

The mitochondria of liver of Yoshida ascites tumour-bearing rats contained two forms of protein kinase distinguishable on the basis of their kinetic properties, substrate specificity and responses to cyclic adenosine 3',5'-monophosphate (cAMP). One of these (kinase I) was activated 2-3 fold by cAMP while the other form (kinase II) was insensitive to the action of cAMP. Kinase I which was selective towards histone F1 as substrate was obtained as a homogeneous preparation and was observed to have a molecular weight of 170 000 by Sephadex G-150 gel filtration. Protein kinase II appeared to be a smaller protein with molecular weight of 54 000 and was specific towards acidic proteins namely casein and phosvitin. Protein kinases isolated from liver mitochondria of normal rats showed variations in respect to elution profile of DEAE-cellulose and electrophoretic mobility. The preparation corresponding to kinase I did not show stimulatory responses to cAMP.     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

Rapid and efficient separation and identification of the two molecular forms of human adenosine deaminase by thin-layer gel filtration chromatography

Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method.     

Fragmentation of mitochondrial H+-ATPase hydrophobic complex]

The subunits with molecular weights of 30 000, 10 000 and 20 000 + 19 000 have been obtained by fractionation of the hydrophobic part of the oligomycin-sensitive ATPase complex on Sephadex G-200 and Sephadex G-150 in the presence of sodium dodecyl sulfate.     

Adenosine deaminase from pig thyroid gland purification and some properties

Adenosine deaminase was isolated from the pig thyroid gland and purified over 900-fold using DEAE Sephadex A-50 column chromatography, Sephadex G-100 gel filtration and DEAE Sephadex A-50 rechromatography. The enzyme was specific towards adenosine. The Michaelis constant based on the Lineweaver-Burk plot was 5 × 10^?5M. The optimum pH was about 7.0, and molecular weight 44 700.     

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.     

Humic-like substances of bacterial origin. II. Fractionation of the bacterial humic-like substances by gel filtration on sephadex gels.

Humic-like substances obtained from cells of Pseudomonas acidovorans were separated on Sephadex G-25 into two groups of substances of different molecular weight. The substances of the molecular weight greater than 5000 were successively separated on Sephadex gels G-50, G-75, G-100. Five fractions of different molecular weight were obtained, the percentage of which varied depending on the media used and time of incubation of the bacteria. Most (38%--46%) of the compounds contained in the bacterial humic acids were of approximate molecular weight of 40 000--50 000. The distribution of the fractions in the bacterial "humic-acids" was compared with those of the humic acid made by Fluka A. G. The synthetic humic acid contained most (approximately 40%) of the compounds of approximate molecular weight of 8000--10 000. In the bacterial and synthetic material the content of the compounds with the molecular weight above 100 000 was very similar (8%--12%).     

Purification and properties of two ribonucleases and a nuclease from barley seeds

Three enzymes possessing RNAase activity were isolated from barley seeds. These enzymes were further purified by ammonium sulphate precipitation DEAE-cellulose chromatography, gel filtration on Sephadex G-75 and DEAE-Sephadex A-50 chromatography. These enzymes have been characterized and classified as: 1. Plant RNAase I (EC 3.1.27.1). It has a pH optimum at 5.7 and molecular weight of 19 000. 2. Plant RNAase II (EC 3.1.27.1). It has a pH optimum at 6.35 and molecular weight of 19 000. 3. Plant nuclease I (EC 3.1.30.2). It has a pH optimum at 6.8 and molecular weight of 37 000. Two RNAases were purified to homogeneity by means of affinity chromatography on poly(G)-Sepharose 4B, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.     

Adenosine kinase from human liver

Adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to homogeneity from human liver. The yield was 55% of the initial activity with a final specific activity of 6.3 mumol/min per mg protein. The molecular weight was estimated as about 40 000 by Sephadex G-100 gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 18% of that of adenosine. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad optimum at pH 7.5--8.5. The Km value for adenosine was 0.15 micrometer, and the activity was strongly inhibited at higher concentrations than 0.5 micrometer. ATP, dATP, GTP and dGTP were proved to be effective phosphate donors. Co2+ was more effective than Mg2+, and Ca2+, Mn2+, Fe2+ and Ni2+ showed about 50% of the activity for Mg2+. Some difference in structure between the adenosine kinase from human liver and that from rabbit or rat tissue, was observed by amino acid analysis and peptide mapping analysis.     

Multiplicity of bovine liver GM1 ganglioside beta-galactosidase

The multiplicity of bovine liver acid beta-galactosidase was investigated. Acid beta-galactosidase activity was measured in the presence of glucono-delta-lactone, which inhibited the neutral beta-galactosidase activity but not the acid beta-galactosidase activity in bovine liver. Three forms of acid beta-galactosidase were separated by Sephadex G-200 gel filtration and the elution pattern of the 4-methylumbelliferyl-beta-galactosidase activity coincided with that of the GM1-beta-galactosidase activity. These forms were relatively stable under acidic conditions (pH 4.5), but the two high molecular weight forms were inclined to dissociate into the low molecular weight form under neutral conditions (pH 7.0). The three forms of the enzyme showed similar pH-optima and apparent Michaelis constants for GM1 ganglioside.     

Purification and properties of rat stomach kallikrein

Kallikrein (EC 3.4.21.8) was purified from rat stomach by column chromatography on p-aminobenzamidine-Sepharose, DEAE-Sephadex A-50 and Sephadex G-150 and by isoelectric focusing, measuring its activities to hydrolyse L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide and to release kinin from heat-treated rat plasma. the purified stomach kallikrein showed a single band on polyacrylamide gel electrophoresis at pH 7.0. Its molecular weight was calculated to be 29 000 by gel-filtration on a column of Sephadex G-50. The kallikrein was stable between pH 6-11 and hydrolyzed L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide optimally at pH 11.0. The L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide hydrolyzing activity of rat stomach kallikrein was inhibited by diisopropyl fluorophosphate and Trasylol, but not by trypsin inhibitors from soybean, lima bean and ovomucoid. These properties of rat stomach kallikrein are different from those of partially purified rat plasma kallikrein, but similar to those of glandular kallikreins from other species. From these results, it was concluded that kallikrein is present in rat stomach and that it can be classified as a glandular kallikrein.     

Adenosine deaminase from Azotobacter vinelandii. Purification and properties

Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65°C, fractionation with ammonium sulfate, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Purified adenosine deaminase was effectively stabilized by the addition of ethylene glycol. The molecular weight of the enzyme was estimated to be 66,000 by gel filtration on Sephadex G-150. The enzyme specifically attacked adenosine and 2-deoxyadenosine to the same extent, and formycin A to a lesser extent. The pH optimum of the enzyme was observed at pH 7.2. Double reciprocal plot of initial velocity versus adenosine concentration was concave upward, and Hill interaction coefficient was calculated to be 1.5, suggesting the allosteric binding of the substrate. ATP inhibited adenosine deaminase in an allosteric manner, whereas other nucleotides were without effect. The physiological significance of the enzyme was discussed in relation to salvage pathway of purine nucleotides.     

Preparation and properties of a new DNase from Aspergillus oryzae.

A DNase present in commercial preparations of Aspergillus oryzae alpha-amylase was purified 1550-fold in 25% yield by acetone precipitation and by chromatography on diethylaminoethyl- and carboxymethylcellulose. The enzyme was isolated free of contaminating RNases and DNases. The molecular weight of the enzyme determined by gel filtration on Sephadex G-100 was 48 000, while a molecular weight of 58 000 was determined for the single band observed upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the DNase is 9.2. The enzyme hydrolyzed only DNA with a pH optimum of 8.2 and was activated by Co2+, and to a lesser extent by Mg2+ and Mn2+. Native DNA was a better substrate than heat-denatured DNA. Enzymatic digests of calf thymus and E. coli DNA yielded oligomers of chain lengths ranging from 10 to 200, with mono- and small oligonucleotides (chain length less than 5) detected only when large (100 mg) amounts of DNA were fractionated by column chromatography on diethylaminoethyl-Sephadex A-25 in 7 M urea. The digestion products contained 5'-terminal phosphate groups and mostly adenosine at the 3' and guanosine and adenosine at the 5' ends.