Low molecular weight angiotensin I converting enzyme from rat lung.

A low molecular weight angiotensin I converting enzyme (light angiotensin enzyme) was isolated from a homogenate of rat lung subjected to dialysis against sodium acetate at pH 4.8. This enzyme has a molecular weight of 84 000 on Sephadex G-200 and a molecular weight of 91 000 on SDS-poly-acrylamide gel as compared with a molecular weight of 139 000 for angiotensin I converting enzyme on SDS-polyacrylamide. Light angiotensin enzyme was activated by NaCl and inhibited by EDTA, angiotensin II, and bradykinin potentiating factor nonapeptide. Light angiotensin enzyme cross-reacted with antibody prepared against angiotensin I converting enzyme and stained with periodic acid-Schiff reagent as a glycoprotein. The evidence suggests that light angiotensin enzyme is a fragment of the higher molecular weight enzyme.     

A new purification procedure for bovine milk xanthine oxidase: effect of proteolysis on the subunit structure.

A new purification procedure for bovine milk xanthine oxidase is reported. The enzyme so obtained is of the highest purity and shows little evidence of degradation. The enzyme displays a single protein band on either polyacrylamide gels or on sodium dodecyl sulfate-urea polyacrylamide gels. Sedimentation equilibrium studies indicate a native molecular weight of 303,000 and a subunit molecular weight of approximately 150,000. The latter value is in good agreement with the minimum molecular weight of 157,000 calculated from dry weight determination and flavin analysis. In contrast, purification of xanthine oxidase from pancreatin-treated cream yields a protein which displays two subunits corresponding to molecular weights of 92,000 and 39,000 as determined by dodecyl sulfate-urea polyacrylamide gel electrophoresis. Pancreatinized enzyme has a greater mobility than unproteolyzed enzyme on polyacrylamide gels. Exposure of milk xanthine oxidase to pancreatin before isolation or after purification yields the same result.     

IMP-cyclohydrolase/transformylase from ehrlich-ascites carcinoma (author's transl)]

The IMP-cyclohydrolase/transformylase enzyme was purified from Ehrlich ascites tumor cells by ammonium sulfate fractionation and chromatography on Sephadex G-75 and DEAE-Sephadex. The electrophoretically pure enzyme has a molecular weight of about 350000, estimated by a gel filtration, and an isoelectric point of 6.2. It is composed of 8 subunits with a molecular weight of 46000. Every subunit is composed of two different proteins with a molecular weight of 18000 and 28500. Some further characteristics of the enzyme are reported.     

Polypeptides from maize seedlings with protein kinase functions

Low molecular weight protein kinase from maize seedlings was isolated. The molecular weight of the enzyme was below 10 000. The enzyme preferentially utilized ATP and casein as donor and acceptor of phosphorus, respectively. Casein kinase was a cyclic nucleotide-independent, heparin-sensitive enzyme. The low molecular weight casein kinase was resolved into basic, neutral and slightly acidic peaks of activity by chromatofocussing.     

Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle

A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3'-dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000-dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits.     

Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. Purification and characterization

NAD+-dependent phenylalanine dehydrogenases were purified 1,500- and 1,600-fold, and crystallized from Sporosarcina ureae SCRC-R04 and Bacillus sphaericus SCRC-R79a, respectively. The purified enzymes were homogeneous as judged by disc gel electrophoresis. The enzyme from S. ureae has a molecular weight of 305,000, while that of B. sphaericus has a molecular weight of 340,000. Each is probably composed of eight subunits identical in molecular weight. The S. ureae enzyme showed a high substrate specificity in the oxidative deamination reaction acting on L-phenylalanine, while that of B. sphaericus acted on L-phenylalanine and L-tyrosine. The enzymes had lower substrate specificities in the reductive amination reaction acting on alpha-keto acids. The Sporosarcina enzyme acted on phenylpyruvate, alpha-ketocaproate, alpha-keto-gamma-methylthiobutyrate and rho-hydroxyphenylpyruvate. The Bacillus enzyme acted on rho-hydroxyphenylpyruvate, phenylpyruvate, and alpha-keto-gamma-methylthiobutyrate. The enzyme from B. sphaericus catalyzes The enzyme from B. sphaericus catalyzes the transfer of pro-S (B) hydrogen from NADH.     

Isolation and characterization of a manganese-containing superoxide dismutase from rat liver.

A manganese-containing superoxide dismutase has been purified from rat liver and characterized. The enzyme has a molecular weight of 89,000 and is composed of four subunits. One atom of manganese is contained per subunit. The metal content, molecular weight, and amino acid analyses show that the rat enzyme is similar to the manganosuperoxide dismutase isolated from human liver.     

Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME

Polyphenol oxidase activity (E.C. 1.14.18.1) has been found in two enzyme species isolated from thylakoid membranes of spinach chloroplasts. The proteins were released from the membrane by sonication and purified >900-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The enzymes appear to be the tetramer and monomer of a subunit with a molecular weight of 42,500 as determined by lithium dodecyl sulfate gel electrophoresis. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.     

The role of phosphatidylglycerol in the activation of CTP:phosphocholine cytidylyltransferase from rat lung.

The reaction catalyzed by CTP:phosphocholine cytidylyltransferase in the reverse direction, i.e. the formation of CTP and phosphocholine from CDP-choline and pyrophosphate, is slightly faster than the reaction in the forward direction. The reverse reaction is optimal at 2 mM pyrophosphate and 6 mM Mg2+, in both fetal and adult preparations. The apparent substrate Km values for phosphocholine, CDP-choline, and pyrophosphate are similar in the fetal and adult forms of the enzyme. The enzyme activity is separated into two forms by gel filtration. The enzyme from adult lung exists as a high molecular weight species, ranging in size from 5 X 10(6) to 50 X 10(6). The enzyme from fetal lung exists as a 190,000 molecular weight species and is totally dependent upon added anionic phospholipid for activity in both the forward and reverse direction. The addition of phosphatidylglycerol gives maximal activity, while phosphatidylinositol or cardiolipin produce about 60 to 70% of the maximal activity. Enzyme activation is accompanied by an aggregation of the enzyme. A sonicated preparation of phosphatidylglycerol is a more efficient activator than a preparation mixed on a Vortex mixer (KA = 30 micronM) and also converts a larger proportion of enzyme from fetal lung into a high molecular weight species. The enzyme from adult lung can be dissociated into a form in fetal lung. The dissociated species can be converted back to a high molecular weight form in the presence of phosphatidylglycerol.     

alpha-galactosidase A from human placenta. Stability and subunit size.

alpha-Galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified from human placenta. The purified enzyme showed one major band on polyacrylamide gel electrophoresis and a single precipitin line on double immunodiffusion. Electrophoresis of the purified, S-carboxymethylated enzyme on sodium dodecyl sulfate polyacrylamide gel showed one component with a molecular weight of about 65 000, but electrophoresis of the non-S-carboxymethylated enzyme showed two components, a major band with a molecular weight of 67 500 and a diffuse band with a molecular weight of 47 000. We suggest that the smaller diffuse component is a degradation product and that the enzyme is a dimer with a molecular weight of approximately 150 000 and a subunit of molecular weight of about 67 500. Antibody raised against the purified enzyme quantitatively precipitated alpha-galactosidase A, but not alpha-galactosidase in Fabry's disease fibroblasts. The alpha-galactosidase A is very heat labile and pH sensitive. It is most stable in concentrated solution at low temperature and at a pH of 5.0 to 6.0. When added to plasma at 37 degrees C, it has a half-life of only 17 min. This imposes a serious obstacle to its use in the treatment of Fabry's disease.     

Identification of pyruvate in S-adenosylmethionine decarboxylase from rat liver

S-Adenosylmethionine decarboxylase has been purified to homogeneity (26,000-fold) from rat liver. The enzyme has a molecular weight of 155,000 and a subunit molecular weight of 42,000. One mole of covalently bound pyruvate was found to be present per mole of enzyme subunit. This is the first mammalian enzyme found to contain covalently linked pyruvate.     

Pyrophosphorylases in Solanum tuberosum: III. PURIFICATION, PHYSICAL, AND CATALYTIC PROPERTIES OF ADPGLUCOSE PYROPHOSPHORYLASE IN POTATOES

ADPglucose pyrophosphorylase from potato (Solanum tuberosum L.) tubers has been purified by hydrophobic chromatography on 3 aminopropyl-sepharose (Seph-C₃-NH₂). The purified preparation showed two closely associated protein-staining bands that coincided with enzyme activity stains. Only one major protein staining band was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The subunit molecular weight was determined to be 50,000. The molecular weight of the native enzyme was determined to be 200,000. The enzyme appeared to be a tetramer consisting of subunits of the same molecular weight. The subunit molecular weight of the enzyme is compared with previously reported subunit molecular weights of ADPglucose pyrophosphorylases from spinach leaf, maize endosperm, and various bacteria. ADPglucose synthesis from ATP and glucose 1-P is almost completely dependent on the presence of 3-P-glycerate and is inhibited by inorganic phosphate. The kinetic constants for the substrates and Mg²⁺ are reported. The enzyme V_max is stimulated about 1.5- to 3-fold by 3 millimolar DTT. The significance of the activation by 3-P-glycerate and inhibition by inorganic phosphate ADPglucose synthesis catalyzed by the potato tuber enzyme is discussed.     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

Dissociation and reassociation of a pig heart phosphoprotein phosphatase.

A pig heart phosphoprotein phosphatase with a molecular weight of 224,000 was dissociated in the presence of 40 % ethanol into an active component (C) of molecular weight 31,000 and components (R) of higher molecular weight. After removal of the ethanol, C and R reassociated and formed an enzyme of molecular weight 188,000. C alone could not form the enzyme. The newly formed enzyme had substrate specificity and response to Mg acetate similar to those of the original large form of the enzyme and was clearly distinguishable from C. The ability of R to associate with C was supressed by treatment of R with trypsin or heat (60°C, 2 min), but not with RNase or DNase.     

Serine transhydroxymethylase isoenzymes from a facultative methylotroph.

Two serine transhydroxymethylase activities have been purified from a facultative methylotrophic bacterium. One enzyme predominates when the organism is grown on methane or methanol as the sole carbon and energy source, whereas the second enzyme is the major isoenzyme found when succinate is used as the sole carbon and energy source. The enzyme from methanol-grown cells is activated by glyoxylate, is not stimulated by Mg2+, Mn2+, or Zn2+, and has four subunits of 50,000 molecular weight each. The enzyme from succinate-grown cells is not activated by glyoxylate and is stimulated by Mg2+, Mn2+, and Zn2+, and sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that this enzyme has subunit molecular weight of 100,000, the same as the molecular weight obtained for the active enzyme. Cells grown in the presence of both methanol and succinate incorporate less methanol carbon per unit time than cells grown on methanol and have a lower specific activity of the glyoxylate-activated enzyme than methanol-grown cells. Adenine, glyoxylate, or trimethoprim in the growth medium causes an increased level of serine transhydroxymethylase in both methanol- and succinate-grown cells by stimulating the synthesis of the glyoxylate-activated enzyme.     

Purification and properties of thiamine pyrophosphokinase in Paracoccus denitrificans

The existence of thiamine pyrophosphokinase [EC 2.7.6.2] in procaryotic cells was first demonstrated in Paracoccus denitrificans (J. Bacteriol, (1976) 126, 1030-1036). The enzyme was therefore purified from this organism to determine its molecular structure and properties. Thiamine pyrophosphokinase which was purified 620-fold from P. denitrificans showed a single band on both polyacrylamide and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular weight in the latter case was calculated to be 23,000. Gel filtration analysis using Sephadex G-150 gave a molecular weight of 44,000, indicating that this enzyme contains at least two identical subunits. Although sedimentation equilibrium analysis gave a molecular weight of 96,000, indirect evidence suggests that the form having this molecular weight is an aggregate of the functional dimer. The activity of the purified enzyme required thiamine, ATP, and Mg2+, and the enzyme catalyzed thepyrophosphorylation of thiamine by ATP. Km values for thiamine and ATP were 10 microM and 0.38 mM, respectively. The activity was competitively inhibited by pyrithiamine, giving a Ki value of 19 microM. Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme. The activity was also inhibited by the product, TPP.     

Biosynthesis of bacterial glycogen: purification and structural properties of Rhodospirillum tenue adenosine diphosphate glucose synthetase.

Adenosine diphosphate glucose synthetase from the photosynthetic bacterium Rhodospirillum tenue has been purified greater than 95%. The molecular weight of the enzyme is approximately 215,000, with a subunit molecular weight of about 51,000. The enzyme appears to be composed of four similar if not identical subunits. Although the amino acid composition of the enzyme is similar to that of Escherichia coli and Salmonella typhimurium, no apparent homology has been observed between their N-terminal amino acid sequences. Antisera prepared against the R. tenue enzyme can partially inhibit the activities of adenosine diphosphate glucose synthetases from other photosynthetic bacteria.     

Aminoacetone oxidase from goat liver. Formation of methylglyoxal from aminoacetone

An enzyme which oxidizes aminoacetone to methylglyoxal has been purified from the particulate fraction of goat liver. Polyamines, such as spermidine and spermine, are also good substrates for this enzyme. The pH optimum for aminoacetone oxidation was found to be 8.2. The apparent Km values of the enzyme for aminoacetone and spermidine were 0.009 and 0.095 mM, respectively. The subunit molecular weight of the enzyme was 93,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the native enzyme was 186,000 by gel filtration. The enzyme is highly sensitive to carbonyl group reagents. The enzyme is not inhibited by monoamine and diamine oxidase inhibitors.     

AMP deaminase from baker's yeast. Kinetic and molecular properties.

The kinetic and molecular properties of AMP deaminase [AMP aminohydrolase, EC 3.5.4.6] purified from baker's yeast (saccharomyces cerevisiae) were investigated. The enzyme was activated by ATP and dATP, but inhibited by Pi and GTP in an allosteric manner. Alkali metal ions and alkaline earth metal ions activated the enzyme to various extent. Kinetic negative cooperativity was observed in the binding of nucleoside triphosphates. Kinetic analysis showed that the number of interaction sites for AMP (substrate) and Pi (inhibitor) is two each per enzyme molecule. The molecular weight of the native enzyme was estimated to be 360,000 by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme gave a single polypeptide band with a molecular weight of 83,000, suggesting that the native enzyme has a tetrameric structure. Baker's yeast AMP deaminase was concluded to consist of two "promoter" units which each consist of two polypeptide chains with identical molecular weight.