Characterisation of cyclic adenosine 3':5'-monophosphate phospodiesterase from Walker carcinoma sensitive and resistant to bifunctional alkylating agents.

Walker carcinoma cell lines sensitive or resistant to bifunctional alkylating agents have been found to contain multiple forms of cyclic AMP phosphodiesterase (3':5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). These activities have been resolved using Sepharose 6B gel filtration and their apparent molecular weights have been estimated. The enzyme appears to occur in four active forms of apparent mol. wts of greater than 1 000 000, 430 000, 350 000 and 225 000, when assayed at low substrate concentrations. Evidence has been obtained which suggests that all four forms of the enzyme are composed of subunits of mol. wt of approximately 15 000 and are interconvertible. While the ionic strength of the buffer affected the predominance of the different forms, the presence of cyclic AMP at 10(-6) M had no effect on aggregation or dissociation of the enzyme. An activity shift from high molecular weight forms of the enzyme to low molecular weight forms has been found in the resistant tumour at low substrate concentration. No change in elution profile between sensitive and resistant tumours was observed for the low affinity form of the enzyme. The pH optima of the enzymes with both high and low affinity for the substrate was found to be pH 8.0 in the sensitive line. In the resistant tumour the pH optima of the high affinity form is shifted to pH 8.4 while the low affinity form remains at pH 8.0. The high affinity forms of the phosphodiesterase in the sensitive and resistant tumour also differed in their inhibition by theophylline. In both cases inhibition was of the competitive type with Ki values for the sensitive and resistant lines being 2.35 and 0.32 mM, respectively. There was no significant difference in the inhibition of the low affinity form between the sensitive and resistant tumour.     

1, 4-alpha-Glucan phosphorylase from Klebsiella pneumoniae purification, subunit structure and amino acid composition.

1. A 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae has been purified about 80-fold with an over-all yield greater than 35%. The purified enzyme has been shown to be homogeneous by gel electrophoresis at different pH-values, by isoelectric focusing, by dodecylsulfate electrophoresis and by ultracentrifugation. 2. The molecular weight of the native enzyme has been determined to be 180 000 by ultra-centrifugation studies, in good agreement with the value of 189 000 estimated by gel permeation chromatography. 3. The enzyme dissociates in the presence of 0.1% dodecylsulfate or 5 M guanidine hydrochloride into polypeptide chains. The molecular weight of these polypeptide chains has been found to be 88 000 by dodecylsulfate polyacrylamide gel electrophoresis and 99 000 by sedimentation equilibrium studies, indicating that the native enzyme is composed of two polypeptide chains. 4. The enzyme contains pyridoxalphosphate with a stoichiometry of two moles per 180 000 g protein, confirming that the 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is a dimeric enzyme. 5. The amino acid composition of the enzyme has been determined, and its correspondence to that of 1,4-alpha-glucan phosphorylases from other sources is discussed. 6. The pI of the enzyme has been shown to be 5.3 and its pH-optimum to be about pH 5.9. The enzyme is stable in the range from pH 5.9 to 10.5.     

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.     

Isolation, purification and physicochemical properties of glutamin-asparaginase from Pseudomonas boreopolis 526

A new homogeneous enzyme which is capable of catalyzing the hydrolysis of both glutamine and asparaginase has been purified from extracts of Pseudomonas boreopolis 526 by the improved method. Purification involves few stages. The ratio of glutaminase to asparaginase activity is approximately 1.5:1.0. The enzyme is stable on storage and has a wide pH optimum of action (6-8.5). The molecular weight is about 134 000-145 000 D and the subunit molecular weight is about 34 000 D. No free SH-groups have been detected in the enzyme molecule.     

Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Plant microbody proteins, I. Purification and characterization of catalase from leaves of lens culinaris.

1) Catalase from green leaves of Lens culinaris (lentils) was investigated with respect to isoenzyme patterns. In contrast to other plants, which have been reported to contain multiple forms of catalase, only one form of this enzyme was revealed when crude extracts were subjected to starch gel electrophoresis or to polyacrylamide disc-gel electrophoresis. Furthermore, catalases from leaves, stems and cotyledons were electrophoretically identical. 2) The leaf enzyme has been purified by conventional methods to apparent homogeneity. It has a molecular weight of 225 000 (ultracentrifuge) and is composed of four identical subunits of molecular weight 54 000 (sodium dodecylsulphate gel electrophoresis). The ratio A280/A405 of the pure enzyme was found to be 1.5. The isoelectric point is at pH 5.5. The enzyme, very labile at pH-values below 7.0, is stable in Tris chloride and potassium phosphate buffers between pH 7.5 and 9.5. It is slowly inactivated by 1mM dithiothreitol and is rapidly inactivated by 1mM mercaptoethanol. 3) The catalase was shown to be the major protein component of the peroxisomal matrix. It could not be detected at the membranes of the leaf peroxisomes.     

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor o.

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission. Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights 0f 32 000, 23 000 and 15 000. The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF1-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark difference in labeling are observed in CF1-depleted chloroplasts which have been treated with N,N'-dicyclohexylcarbodiimide. The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CFo) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CFo, and the light/dark labeling differences may indicate conformational changes within CFo. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

Enzymes and cancer: Preparation and some properties of guanase from rabbit liver

1. Guanase has been purified 200-fold in 20% yield from the supernatant fraction of rabbit-liver homogenates, by using ammonium sulphate fractionation, calcium phosphate-gel adsorption and chromatography on DEAE-cellulose and Sephadex G-200. 2. K_m with guanine as substrate at the optimum pH of 7·7 was found to be 1·05×10⁻⁵m. Q₁₀ was 1·4 between 23° and 48°. 3. Substrate activity and pH optima of compounds related to guanine have been studied. 8-Azaguanine, 1-methylguanine, thioguanine and 1-methylthioguanine are all substrates.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.     

Acid inactivation of and incorporation of phosphate into alkaline phosphatase from Escherichia coli

1. Alkaline phosphatase of Escherichia coli undergoes below pH 6·0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6·0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3·5 is rapidly recovered when the enzyme is taken back to pH 8·0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn²⁺, although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme.     

Partial characterization of 2 extracellular aminopeptidases (arylamidases) from the dermatophyte Keratinomyces ajelloi]

Two aminopeptidases AP1 and AP2 have been isolated from Keratinomyces ajelloi filtrates. The molecular weight is about 27 000 for AP1 and 23 000 for AP2. Both aminopeptidases present maximum activity at pH 9.35 but 50 p. 100 of maximum activity is observed between pH 7.5 and pH 8.5. Km values measured at pH 9.35 with L-leucine-p-nitroanilide as substrate are 0.38 X 10(-3) M for AP1 and 0.43 X 10(-3) M for AP2. kcat at the same pH are 63.6 sec.-1 for AP-1 and 62.8 sec-1 for AP2. Both aminopeptidases are inhibited by mercuric chloride, o-phenanthroline, dithiothreitol and 2-mercaptoethanol. Some of their characters make them similar to Streptomyces griseus pronase aminopeptidases.     

Some characteristics of sn-glycero-3-phosphocholine diesterases from rat brain.

1. sn-Glycero-3-phosphocholine diesterase activities, glycerophosphohydrolase (EC 3.1.4.2) and choline phosphohydrolase (EC 3.1.4.38) from rat brain have been partially purified and characterized using sn-glycere-3-[32P]phosphocholine as substrate and separating the reaction products by anion-exchange chromatography and ionophoresis. 2. Rat brain contained particulate (75%) and soluble (25%) activity from both diesterases. No difference in pH optimum or metal ion requirement for the particulate compared to the soluble enzymes was observed. 3. Glycerophosphohydrolase (EC 3.1.4.2) was purified 60-fold, choline phosphohydrolase (EC E.1.4.38) 120-fold from rat brain supernatant fraction by DEAE-cellulose ion-exchange chromatography and sucrose density gradient centrifugation. The density gradient results in conjunction with dodecyl sulphate-polyacrylamide gel disc electrophoresis yielded molecular weight estimates of 230 000 (monomer 62 000) for choline phosphohydrolase and 120 000 (monomer 70 000) for glycerophosphohydrolase (EC 3.1.4.2). 4. Glycerophosphohydrolase (EC 3.1.4.2) has a pH optimum of 8.9 and a Km for sn-glycero-3-phosphocholine of 0.6 mM. The enzyme is inhibited by EDTA and reactivated by Ca2+. Choline phosphohydrolase (EC 3.1.4.38) has pH optimum 10.5, a Km of 2 mM and is unaffected by EDTA. Both enzymes require Ca2+ for maximum activity.     

Spectroscopic characterization of the Stentor photoreceptor.

1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.     

Characterization of the active site of homogeneous thyroid purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (purine-nucleoside : orthophosphate ribosyltransferase, EC 2.4.2.1) has been purified approx. 4000-fold and to electrophoretic homogeneity from bovine thyroid glands. The isolated enzyme has a specific activity of 17 mumol . min-1 . mg-1. The native enzyme appears to have a molecular weight of 92 000 as determined by sedimentation equilibrum ultracentrifugation and is comprised of three subunits having a molecular weight of 31 000 each as shown by sodium dodecyl sulfate gel electrophoresis. The enzyme is irreversibly denatured below pH 5 and the enzyme-substrate complex is shown to have an ionization constant (pKa) of 9.2 which influences catalytic activity. The pH dependence of the kinetic constants identifies three amino acid ionizable protons. The binding of inosine is effected by an imidazole ring of histidine (pKa 5.65) and a sulfhydryl group of cysteine (pKa 8.5) and the maximal velocity is restricted by an epsilon-amino group which is essential for phosphate binding. The requirement of these residues for activity was confirmed by group-specific chemical modification. The presence of phosphate protected only the lysyl residue while inosine protected all three residues from chemical titration. A model is proposed for the catalytic mechanism of purine nucleoside phosphorylase.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The purification and properties of histidinol dehydrogenase from Neurospora crassa

1. A procedure is described for the purification of l-histidinol dehydrogenase (l-histidinol-NAD oxidoreductase, EC 1.1.1.23) from Neurospora crassa. 2. The enzyme, as purified, has a sedimentation coefficient, S(20), of 7.1s and a molecular weight of 81 000. Considerable variation is possible in the state of polymerization of the enzyme, giving rise to observed molecular weights from 40 000 to 240 000. 3. Several kinetic parameters of the enzyme have been determined. The enzyme is maximally active at pH9.8; the K(m) (NAD) is 13.0x10(-5)m and K(m) (histidinol) is 8.2x10(-6)m. The enzyme is highly specific, does not oxidize a range of amino alcohols and other aliphatic alcohols nor reduce NADP and has no demonstrable affinity for histidine. The turnover number is 49 moles of NAD reduced/min./mole of enzyme (mol.wt. 40 000).     

Light/dark labeling differences in chloroplast membrane polypeptides associated with chloroplast coupling factor 0

The fluorogenic reagent fluorescamine has been used to determine the labeling patterns of Type C spinach chloroplast membrane polypeptides. Membrane polypeptides labeled with fluorescamine were detected by scanning high resolution sodium dodecyl sulfate polyacrylamide gradient slab gels for fluorescence emission.Three membrane polypeptides show a decrease in the extent of labeling when chloroplast membranes are labeled in the light compared to when they are labeled in the dark. These polypeptides have apparent molecular weights of 32 000, 23 000 and 15 000.The decrease in labeling observed in the light is abolished or reduced by treatments which inactivate the light-generated transmembrane pH gradient. CF₁-depleted chloroplasts show neither a light-activated pH gradient nor a light/dark difference in labeling of these three polypeptides. Both a light-activated pH gradient and light/dark differences in labeling are observed in CF₁-depleted chloroplasts which have been treated with N,N′-dicyclohexylcarbodiimide.The same ammonium sulfate fractions of a 2% sodium cholate extract, which are believed to be enriched in the membrane-bound sector of the chloroplast ATPase (CF_o) are also found to be enriched in the 32 000, 23 000 and 15 000 molecular weight polypeptides. The three polypeptides are believed to be components of CF_o, and the light/dark labeling differences may indicate conformational changes within CF_o. Such conformational changes may reflect a mechanism which couples light-generated proton gradients to ATP synthesis.     

Stability of 2'-deoxyxanthosine in DNA

The deamination of nucleobases in DNA occurs by a variety of mechanisms and results in the formation of hypoxanthine from adenine, uracil from cytosine, and xanthine and oxanine from guanine. 2′-Deoxyxanthosine (dX) has been assumed to be an unstable lesion in cells, yet no study has been performed under biological conditions. We now report that dX is a relatively stable lesion at pH 7, 37°C and 110 mM ionic strength, with a half-life (t_1/2) of 2.4 years in double-stranded DNA. The stability of dX as a 2′-deoxynucleoside (t_1/2 = 3.7 min at pH 2; 1104 h at pH 6) was increased substantially upon incorporation into a single-stranded oligodeoxynucleotide, in which the half-life of dX at different pH values was found to range from 7.7 h at pH 2 to 17 700 h at pH 7. Incorporation of dX into a double-stranded oligodeoxynucleotide resulted in a statistically insignificant increase in the half-life to 20 900 h at pH 7. Data for the pH dependence of the stability of dX in single-stranded DNA were used to determine the rate constants for the acid-catalyzed (2.6 × 10^–5 s^–1) and pH-independent (1.4 × 10^–8 s^–1) depurination reactions for dX as well as the dissociation constant for the N⁷ position of dX (6.1 × 10^–4 M). We conclude that dX is a relatively stable lesion that could play a role in deamination-induced mutagenesis.     

Purification and properties of L-asparaginase EC-2 from Escherichia coli 055:B5.

1. L-Asparaginase has been isolated from aerobically grown Escherichia coli 055:B5 and purified about 140-fold in a three-step procedure involving acidification to pH 4.5, ammonium sulphate fractionation and column chromatography on DEAE-Sephadex A-50. The activity of the preparation is 140 U/mg protein. 2. The enzyme acts within a broad pH range (pH 5-9) and is affected neither by PCMB, N-ethylmaleimide nor metal ions. 3. Molecular weight of the isolated asparaginase is 130 000.     

Partition of porphyrins between cyclohexanone and aqueous sodium acetate as a function of pH. Determination of uroporphyrin and of hydrophilic porphyrin conjugates

1. The partition of uroporphyrins I and III, coproporphyrins I and III, haematoporphyrin IX, porphyrin c and a hydrophilic porphyrin–peptide fraction from variegate-porphyria faeces has been studied in systems of equal volumes of cyclohexanone and sodium acetate buffers of varying pH and concentration. 2. The concentration of acetate in the aqueous phase has little effect on the partition of porphyrin c, but markedly influences that of uroporphyrin. At 50% acetate saturation and pH4·5, only 5% enters the cyclohexanone phase whereas 60% of porphyrin c is extracted under similar conditions. 3. This circumstance forms the basis of a method for the determination of hydrophilic porphyrin–peptides in variegate-porphyria urine. Its reliability has been checked in model experiments. 4. At pH1·5 and an aqueous phase half-saturated with sodium acetate, an equal volume of cyclohexanone removes 95–97% of uroporphyrin and about 55% of porphyrin c. Uroporphyrin may therefore be determined as a second step in the method. 5. For the routine determination of uroporphyrin in systems free from other hydrophilic porphyrins, cyclohexanone extraction may be performed at any pH in the range 1·0–3·0.