Purification and characterization of nucleoside diphosphate kinase from spinach leaves.

Two types of nucleoside diphosphate kinase (NDP kinase I and NDP kinase II) have been purified from spinach leaves to electrophoretic homogeneity. The enzymes were copurified with apparent [35S]GTP-gamma S-binding activities. NDP kinase I, which was not adsorbed to a hydroxyapatite column, and NDP kinase II, which was adsorbed, had molecular weights of 16,000 and 18,000, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weights determined by gel filtration were 92,000 and 110,000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor differences in some amino acids between NDP kinase I and NDP kinase II were observed when both enzymes were analyzed for amino acid composition. The apparent [35S]GTP gamma S-binding activity of purified NDP kinase I and NDP kinase II was found to be due to the formation of a [35S]thiophosphorylated enzyme, which is the intermediate of the NDP kinase reaction.     

Phenylalanyl-tRNA synthetase from chloroplasts of a higher plant (Phaseolus vulgaris). Purification and comparison of its structural, functional, and immunological properties with those of the enzymes from the corresponding cytoplasm, the cyanobacterium Anacystis nidulans, and the photosynthetic green sulfur bacterium Chlorobium limicola

Chloroplastic phenylalanyl-tRNA synthetase from bean leaves is purified under optimal protective conditions over 4,900-fold. Its apparent molecular weight is 78,000, as determined by gel filtration, with a dimeric subunit structure of alpha beta (alpha = 33,000 and beta = 42,000), as determined by sodium dodecyl sulfate gel electrophoresis. This indicates a drastic size reduction of 40% for each subunit compared to the corresponding cytoplasmic enzyme and a unique quaternary structure. Heterologous aminoacylation and substrate properties of ATP analogs indicate substantial differences in the topographies of the substrate binding domains of these two heterotopic intracellular plant enzymes. No common antigenic determinants with the bean cytoplasmic enzyme were detected by polyclonal antibodies against the chloroplastic enzyme. The same negative result applies to the immunological comparison with the partially purified enzymes from the cyanobacterium Anacystis nidulans and the photosynthetic green sulfur bacterium Chlorobium limicola that both have a molecular weight of 260,000.     

Two forms of glutamine synthetase from pumpkin leaves

Two molecular forms of glutamine synthetase localized in the cytoplasm and in chloroplasts, respectively, were detected in pumpkin leaves. Ammonium infiltrated into intact pumpkin leaves activated the synthesis of both enzyme forms. Glutamine synthetase from chloroplasts and the cytoplasmic enzyme were purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose DE-32, selective adsorption on potassium phosphate gel and preparative electrophoresis in polyacrylamide gel. The molecular weights of both forms of glutamine synthetase as determined by gel-filtration through Sephacryl S-200 are equal to 370,000 and 480,000, respectively. During SDS polyacrylamide gel electrophoresis the enzymes from both sources produced polypeptide chains with respective molecular weights of 50,000 and 58,000. The amino acid composition of the enzymes differed considerably. The content of alpha-helix moities in the chloroplast and cytoplasmic enzyme made up to 17% and 34%, respectively. In the presence of Mg+ the pH optima for the enzymes were equal to 7.75 and 7.25, respectively, and the Km values for L-glutamate were 46 and 13 mM, respectively. It may be concluded that the enzyme forms under study are isoenzymes.     

Comparison of tRNA nucleotidyltransferase from Escherichia coli and Lactobacillus acidophilus.

Purification of tRNa nucleotidyltransferase from Lactobacillus acidophilus ATCC 4963 and Escherichia coli MRE 600 by preparative polyacrylamide gel electrophoresis is described. Both enzymes gave a single band on analytical polyacrylamide-gel electroesis and sodium dodecylsulfate gels. Chromatography of the high speed supernatant from Lactobacillus at low salt concentrations gave three enzyme fractions of molecular weights about 45 000, 90 000, and 120 000. At 1M NaCl only the first enzyme fraction was found. Kinetic data for both enzymes are given.     

Pathogen-induced vascular gels: Ethylene as a host intermediate

A cell free culture filtrate from 6-day cultures of Fusarium oxysporum f. sp. cubense was processed to give: (1) a heterogeneous enzyme mixture, (2) purified polygalacturonase (PG), (3) partially-purified polygalacturonate lyase and (4) β-1,4-xylanase. When introduced into explanted castor bean leaves each of these preparations was able to promote the formation of vascular system-obstructing gels. Exposure of castor bean leaves to ethylene (3 ppm) also triggered gel formation. Explanted leaves produced ethylene in response to the enzyme mixture and PG. Vascular gel formation did not occur when ethylene production in response to enzymes was prevented.     

Comparative studies on the molecular weight of glutathione S-transferases from mammalian livers and an insect.

1. A comparative study was conducted on the molecular weights of glutathione S-transferases in the housefly and liver of the mouse and rat using Sephadex G-100 gel chromatography. 2. The values varied depending upon the buffers used in gel filtration. Molecular weights of 44,600, 53,600 and 43,000 daltons respectively were obtained with 0.01 M potassium phosphate buffer, pH 6.7; 0.05 M Tris-HCl buffer, pH 8.0; and 0.05 M Tris-HCl buffer containing 0.1 M KCl, pH 8.0, respectively. 3. There was no difference in the molecular weights of the enzymes obtained from the insect and from the mammalian livers. Purified enzymes eluted in the same fractions as those from the crude extracts, suggesting little modification in the molecular size of the enzymes during purification. 4. The presence of a large volume of stabilizer(s) in the enzyme solutions applied to the column delayed the elution of the activity peaks and resulted in erroneous values. Therefore, different literature values of molecular weights for glutathione S-transferases may be the result of different buffers and stabilizers used in gel filtration and probably do not represent a real difference in molecular size.     

The characteristics of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferases from Sansevieria trifasciata leaves and Phaseolus coccineus stems

Three different molecular forms of pyrophosphate-dependent phosphofructokinase have been isolated: one from Sansevieria trifasciata leaves and two from Phaseolus coccineus stems. The form isolated from S. trifasciata has the molecular weight of about 115,000. The apparent molecular weights for the two forms from mung bean were approximately 220,000 and 450,000. All three forms have the same pH optima, an absolute requirement for Mg2+ ions both in the forward and reverse reaction, but differ in their sensitivity toward fructose 2,6-bisphosphate. Kinetic properties of the partially purified enzymes have been investigated in the presence and absence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase from S. trifasciata exhibited hyperbolic kinetics with all substrates tested. The saturation curves of the enzyme (form A) from mung bean for pyrophosphate, fructose 6-phosphate and fructose 1,6-bisphosphate were sigmoidal in the absence of fructose 2,6-bisphosphate. In the presence of fructose 2,6-bisphosphate these kinetics became hyperbolic.     

Purification and characterization of the soluble form of mitochondrial adenosine triphosphatase from sweet potato

The soluble form of mitochondrial adenosine triphosphatase was purified in an electrophoretically and immunologically pure form from sweet potato root tissue. The enzyme consisted of six kinds of subunits with different molecular weights (52,500, 51,500, 35,500, 26,000, 23,000, and 12,000), and its molecular weight was about 370,000. Adenosine triphosphatase associated with the submitochondrial particles was oligomycin-sensitive and heat-labile, whereas the soluble form of the enzyme was oligomycin-insensitive and cold-labile. The enzyme in either the membrane-bound or the soluble form showed negative cooperativity. Both experiments with polyacrylamide gel electrophoresis and immunological methods suggest that some of the subunits, probably those with molecular weights of 52,500 and 51,500, are dissociated from the enzyme protein during storage of the enzyme preparations.     

Light modulation of maize leaf phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH₄)₂SO₄ fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.     

Lipid metabolism in germinating seeds. Purification of ethanolamine kinase from soya bean.

Ethanolamine kinase has been purified to homogeneity from germinating soya bean (Glycine max L.) seeds. The purified enzyme had a molecular weight of 17--19 000 as estimated by gel filtration and sodium dodecyl suphate-polyacrylamide gel electrophoresis. It would not phosphorylate choline, had a Km for ethanolamine of 8 microM and utilised Mg-ATP. The kinase could be purified in a 37 000 molecular weight form (dimer) which would easily dissociate on storage. In contrast to ethanolamine kinase whose activity was unaffected by the presence of choline in the assay system, soya bean choline kinase, although not phosphorylating ethanolamine, was competitively inhibited by the latter. The purification of specific choline and ethanolamine kinases from germinating soya bean confirmed in vivo observations which had indicated separate enzymes.     

Characterization of alpha-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.     

Purification and characterization of an NADP+-linked alcohol oxido-reductase which catalyzes the interconversion of gamma-hydroxybutyrate and succinic semialdehyde.

Abstract— An NADP⁺ -linked enzyme, capable of interconverting γ-hydroxybutyrate and succinic semialdehyde, has been isolated from hamster liver and brain. The enzyme which was isolated from liver has been purified 300-fold and exhibits a single band by polyacrylamide gel electrophoresis. The molecular weight of the enzyme is - 31,000 as estimated from gel filtration and 38,000 as estimated from sodium dodccyl sulfate gel electrophoresis. The enzyme is inhibited by amobarbital, diphenylhy-dantoin, 2-propylvalerate, and diethyldithiocarbamate, but not by pyrazole. The enzymes from brain and liver appear to be very similar with regard to their molecular weights and their kinetic constants for γ-hydroxybutyrate and succinic semialdehyde.     

Purification of monomeric agmatine iminohydrolase from soybean

Agmatine iminohydrolase (EC 3.5.3.12) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by chromatographic separations on Sephadex G-25, Bio-rex 70, and agmatine-affinity columns. The enzyme was homogeneous by the criteria of analytical gel electrophoresis. Molecular weights estimated by Sephadex G-100 gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis were 70,000, indicating that the soybean axes enzyme is a monomer, in contrast to the dimeric enzymes from corn and rice. The isoelectric point determined by gel electrofocusing was 7.5, higher than that of the corn enzyme (4.7). The optimal pH and temperature for activity were 6.5 and 50 degrees C, respectively. The enzyme has high specificity for agmatine, and the Km for agmatine was 2.5 x 10(-3) molar. The enzyme was sensitive to Cu2+ and also was inhibited by p-hydroxymercuribenzoate.     

Purification and physico-chemical properties of glutamine synthetase from pea chloroplasts]

A highly purified, practically homogeneous glutamine synthetase was isolated from pea leaf chloroplasts. The enzyme purity was assayed by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation coefficient is 16,3S. The sedimentation equilibrium analysis showed that the molecular weight of the enzyme is equal to 480 000. The minimal molecular weights of the enzyme as calculated from the data of polyacrylamide gel electrophoresis in the presence of SDS and the amino acid analysis were found to be 62 000 and 60 000, respectively. The enzyme contains a large amount of dicarboxylic and sulfur-containing amino-acids. The N-terminal amino acid is glycine. The isoelectric point for the enzyme lies within the pH range of 4,2-4-4.     

Properties and quaternary structure of ribulosediphosphate carboxylase from the leaves of Elymus (Psathyrosachys) junceus

The enzyme ribulosdiphosphate carboxylase was isilated from the leaves of Elymus (Psathyrosachys) junceus. The enzyme was found homogenous during disc-electrophoresis in polyacrylamide gel and analytical ultracentrifugation. The sedimentation coefficient for the enzyme is 17,4S. The enzyme molecular weight as determined by the sedimentation equilibrium technique is equal to 540000. The enzyme molecule consists of 2 types of subunits, i.e. the larger subunit has m.w. of 55000, the smaller one--12900. The number of large subunits is 8, that of small ones--8. The specific activity of the homogenous enzyme makes up to 2,45 mkmoles of CO2 per min per mg of protin (pH 8,0, 30 degrees). The purified enzyme was stable in Mg2+- and dithiothreitol-containing buffers for 3--4 weeks at 4 degrees and for 5--6 months at --20 degrees. The amino acid composition of the enzyme molecule is similar to that of the enzyme from spinach leaves.     

Purification and properties of glyoxysomal lipase from castor bean

The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes.     

The absence of structural relationship between mitochondrial and mitochondrial and cytoplasmic leucyl-tRNA synthetases from Tetrahymena pyriformis.

Two leucyl-tRNA synthetases (EC 6.1.1.4) have been purified to near homogeneity, the one from mitochondria and the other from cytoplasm of Tetrahymena pyriformis. Both enzymes were found to be structurally unrelated, single polypeptides with molecular weights of approximately 100,000 as determined by gel permeation, sucrose gradient centrifugation, and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These enzymes behaved differently in elution profiles through hydroxyapatite- and diethylaminoethyl cellulose-column chromatography and isoelectric focusing. The two enzymes also showed some differences in responses to various salts for charging and in pH optima and temperature sensitivity, but no significant difference was found in their affinities (K_m) for ATP and leucine. These enzymes recognized different leucyl-tRNA isoaccepting species as revealed by reversed-phase column chromatography. The mitochondrial enzyme can charge six isoaccepting leucyl-tRNA species, while the cytoplasmic enzyme can recognize only four species.     

The estimation and comparison of molecular weight of angiotensin I converting enzyme by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis.

The angiotensin I converting enzyme from rat lung was observed to be a glycoprotein containing 8.3% carbohydrate and consisting of a single polypeptide chain with an estimated molecular weight of 139 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and 150 000 by sucrose density gradient sedimentation analysis. A comparison of the mobility of angiotensin I converting enzyme from rat lung, rabbit lung, and two hog lung sources on sodium dodecyl sulfate-polyacrylamide gels indicates that all four enzymes have very similar molecular weights and subunit structures. Some previously reported molecular weight discrepancies appear to be due to anomalous behavior of the enzyme of gel filtration.     

Purification and Some Properties of Two Aminopeptidases from Sardines

Two fish aminopeptidases designated as aminopeptidases I and II were purified by DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. The final preparations of enzymes I and II were judged nearly homogenous by polyacrylamide gel I, electrophoresis. The molecular weights of enzymes I and II were determined by gel filtration to be 370,000 and 320,000, respectively. The isoelectric points were 4.1 (I) and 4.8 (II), Both enzymes were inhibited by EDTA and activated by Co⁺⁺. Bestatin could inhibit enzyme I but not enzyme II. Enzymes I and II rapidly hydrolyzed not only synthetic substrates containing alanine or leucine but also di-, tri-, and tetra-alanine. Judged from all of these properties, sardine aminopeptidases resemble human alanine aminopeptidase. Enzyme I retained more than 70% of its original activity in 15% NaCl, suggesting the enzyme participates in hydrolyzing fish proteins and peptides during fish sauce production.