scholarly journals Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties

1972 ◽  
Vol 129 (3) ◽  
pp. 571-581 ◽  
Author(s):  
B. L. Ong ◽  
J. F. Jackson
Keyword(s):  
Gel Filtration ◽  
Deae Cellulose ◽  
Aspartate Transcarbamoylase ◽  
Partial Purification ◽  
Saturation Curve ◽  
Phaseolus Aureus ◽  
Phosphate Binding ◽  
Carbamoyl Phosphate ◽  
Sequential Reaction ◽  
Substrate Saturation

1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH4)2SO4 fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required Pi for maximum stability, had an apparent molecular weight of 83000±5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5–10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the Km for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of Pi. Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.

The Separation and Partial Purification of the Soluble 17α- and 17β-Hydroxysteroid Dehydrogenases of Rabbit Liver

1974 ◽  
Vol 52 (2) ◽  
pp. 120-125 ◽  
Author(s):  
Sadiq Hasnain ◽  
Denis G. Williamson
Keyword(s):  
Dehydrogenase Activity ◽  
Enzyme Activities ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Rabbit Liver ◽  
Partial Purification ◽  
17Β Estradiol ◽  
Derivatives Of ◽  
Estradiol 17Β

17-Hydroxysteroid dehydrogenase activity toward 17α-estradiol, 17β-estradiol, and the 3-glucuronide derivatives of these steroids has been demonstrated in the 105 000 × g supernatant of rabbit liver. DEAE-cellulose chromatography of the enzyme activities isolated by calcium phosphate gel fractionation and Sephadex gel filtration yielded a single 17α-hydroxysteroid dehydrogenase activity toward both 17α-estradiol and 17α-estradiol 3-glucuronide. However, separate 17β-hydroxysteroid dehydrogenase activities toward 17β-estradiol and 17β-estradiol 3-glucuronide were obtained. Further purification of the 17α-hydroxysteroid dehydrogenase fraction by isoelectric focusing resulted in multiple peaks of activity toward 17α-estradiol and 17α-estradiol 3-glucuronide. One of these enzyme activities was highly specific for the 3-glucuronide derivative, it being essentially devoid of activity toward 17α-estradiol.

scholarly journals β-Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-β-hydroxybutyrate metabolism

1973 ◽  
Vol 134 (1) ◽  
pp. 239-248 ◽  
Author(s):  
Volker Oeding ◽  
Hans G. Schlegel
Keyword(s):  
Condensation Reaction ◽  
Gradient Centrifugation ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Saturation Curve ◽  
Cleavage Reaction ◽  
Acetyl Coa ◽  
Freeze Dried ◽  
Exclusion Chromatography

1. β-Ketothiolase was purified 49-fold from fructose-grown cells of Hydrogenomonas eutropha H16 with a yield of 27%; the purification procedure involved precipitation by cetyltrimethylammonium bromide, DEAE-cellulose chromatography and exclusion chromatography on Sephadex G-200; the freeze-dried enzyme is stable. The molecular weight determined by sucrose-gradient centrifugation (8.2S) and by gel filtration is 147000–150000. The optimum pH for the cleavage reaction is 8.1, that for the condensation reaction 7.8, both measured in Tris–HCl buffer. 2. The kinetics of the cleavage reaction are described. Substrate-saturation curves were measured with both acetoacetyl-CoA and CoA as the variable substrates. The concentration of the second substrate was kept constant and was varied during successive experiments. The cleavage reaction is characterized by substrate inhibition by acetoacetyl-CoA, which is partially relieved by free CoA. Hill plots indicate two acetoacetyl-CoA-binding sites. 3. The substrate(acetyl-CoA)-saturation curve for the condensation reaction is hyperbolic. The Km was 3.9×10−4m-acetyl-CoA. In the presence of CoA sigmoidal curves were obtained, with an increasing sigmoidicity from 0.03 to 0.30mm-CoA. The inhibitory action of CoA on the β-ketothiolase condensation reaction and its possible involvement in the regulation of poly-β-hydroxybutyrate synthesis and degradation are discussed.

scholarly journals Pyrimidine nucleotide biosynthesis in Phaseolus aureus. Enzymic aspects of the control of carbamoyl phosphate synthesis and utilization

1972 ◽  
Vol 129 (3) ◽  
pp. 583-593 ◽  
Author(s):  
B. L. Ong ◽  
J. F. Jackson
Keyword(s):  
Inhibitory Effect ◽  
Aspartate Transcarbamoylase ◽  
Synthetase Activity ◽  
Carbamoyl Phosphate Synthetase ◽  
Phaseolus Aureus ◽  
Carbamoyl Phosphate ◽  
Purine Nucleotides ◽  
Nucleotide Biosynthesis ◽  
Pyrimidine Nucleotide ◽  
Ornithine Transcarbamoylase

1. Carbamoyl phosphate synthetase activity of Phaseolus aureus extracts was assayed by coupling it to the catalytic subunit of Escherichia coli aspartate transcarbamoylase and determining the [14C]carbamoylaspartate so formed. The stability of the activity was improved by the addition of ornithine and dimethyl sulphoxide to the extraction medium. 2. The synthetase activity was found to utilize either glutamine or ammonia as amino donor, the Michaelis constants being 0.17±0.03mm and 6.1±1.0mm respectively. N-Acetylglutamate did not significantly alter the rate with either substrate, and azaserine inhibited the reaction with both amino donors to the same extent. 3. Ornithine was shown to stimulate the activity, and to counteract inhibition by UMP. The purine nucleotides IMP and GMP enhanced carbamoyl phosphate formation, whereas AMP had an inhibitory effect. 4. The Michaelis constant for carbamoyl phosphate was determined in concentrated extracts for both aspartate transcarbamoylase and ornithine transcarbamoylase activities, and was 0.13±0.03mm and 1.58±0.16mm respectively. The ratio of the activities of these two enzymes, determined at near-saturating substrate concentrations, was 1:3 (aspartate transcarbamoylase/ornithine transcarbamoylase). 5. It is concluded that in this plant tissue there is one enzyme, carbamoyl phosphate synthetase, supplying carbamoyl phosphate to both the pyrimidine and arginine pathways, that the pyrimidine pathway claims most of the available carbamoyl phosphate (depending on the concentration of the nucleotide effectors) when this intermediate is present at low concentrations; and that when the carbamoyl phosphate concentration is increased, possibly by ornithine stimulation, a larger proportion can be taken up by the arginine pathway.

scholarly journals Modulation of guanine deaminase

1973 ◽  
Vol 131 (4) ◽  
pp. 683-687 ◽  
Author(s):  
K. Sree Kumar ◽  
A. Sitaramayya ◽  
P. S. Krishnan
Keyword(s):  
Rat Brain ◽  
Substrate Concentration ◽  
Mouse Liver ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Saturation Curve ◽  
Guanine Deaminase ◽  
Sigmoidal Kinetics ◽  
Substrate Saturation

1. Guanine deaminases purified from the 15000g supernatant fraction of iso-osmotic sucrose homogenates of rat and mouse liver and brain were tested for the influence of GTP and allantoin. 2. The suffixes A and B were assigned to the isoenzyme fractions eluted from DEAE-cellulose with the lower and the higher molarity of eluent respectively. Isoenzyme A from rat liver, the activity of which showed a sigmoid dependence on substrate saturation, was activated by GTP and inhibited by allantoin. Isoenzyme B, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin. 3. Isoenzyme A from rat brain, the activity of which had a sigmoid dependence on substrate concentration, was stimulated by GTP. Isoenzyme B, which showed classical Michaelis–Menten kinetics, was inhibited by allantoin. 4. Mouse liver guanine deaminase was not influenced by either GTP or allantoin. 5. Isoenzyme A from mouse brain, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin but isoenzyme B, with sigmoidal kinetics, was inhibited by allantoin. 6. Mg2+ activated, or inhibited or did not have an effect on guanine deaminase, depending on the source of the enzyme. 7. The bearing of the above findings on the possible regulation of guanine deaminase activity in vivo is discussed.

scholarly journals Mitogenic polypeptide of the mammalian seminiferous epithelium: biochemical characterization and partial purification.

1983 ◽  
Vol 97 (5) ◽  
pp. 1435-1443 ◽  
Author(s):  
L A Feig ◽  
M Klagsbrun ◽  
A R Bellvé
Keyword(s):  
Sertoli Cells ◽  
Adult Mouse ◽  
Biochemical Characterization ◽  
Mammalian Species ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Seminiferous Epithelium ◽  
Partial Purification ◽  
Gel Filtration Chromatography

A mitogenic polypeptide, previously identified in Sertoli cells of the prepuberal mouse (Feig, L. A., A. R. Bellvé, N. Horbach-Erickson, and M. Klagsbrun, 1980, Proc. Natl. Acad. Sci. USA., 77:4774-4778), now has been shown to exist in Sertoli cells of the adult mouse and in the seminiferous epithelium of several other mammalian species, including the rat, guinea pig, and calf. The levels of this seminiferous growth factor (SGF) are not appreciably reduced in adult mouse testes following hypophysectomy. SGF purified from either the adult mouse or newborn calf seminiferous epithelium has a molecular weight (Mr) of 15,700 and a pl between 4.8 and 5.8, when exposed to denaturing conditions. Furthermore, SGF from these two mammalian species probably has few exposed hydrophobic domains and has a strong propensity to aggregate into multiple, high Mr species. A purification sequence based on these biochemical properties has enabled a greater than 350-fold enrichment of SGF activity from the calf seminiferous epithelium. The protocol involves a sequence of: (a) ammonium sulfate precipitation, (b) DEAE-cellulose ion exchange chromatography, (c) gel filtration chromatography on Bio-Gel P150 in 1.0 M ammonium acetate, (d) hydrophobic chromatography on dodecyl agarose, and (e) gel filtration chromatography in 6.0 M guanidine hydrochloride. Subsequent analysis of this purified preparation by SDS PAGE, followed by silver staining, reveals approximately 7 polypeptides with Mr between 14,000 and 20,000.

scholarly journals Partial Purification of Deamidase from Germinating Wheat (Triticum aestivum L.) Seeds and its Application for the Improvement of Functional Properties of Wheat Gluten

1970 ◽  
Vol 15 ◽  
pp. 89-98
Author(s):  
ATM Mijanur Rahman ◽  
NK Sana ◽  
Md Masudul Hasan Khan ◽  
BC Sarkar ◽  
EM Huque ◽  
...  
Keyword(s):  
Functional Properties ◽  
Wheat Gluten ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Foam Formation ◽  
Salting Out ◽  
Key Words Wheat ◽  
Sds Page ◽  
Wheat Seeds

Enzyme with deamidase activity was found in the germinating wheat seeds. The activity appeared in the seeds after 24 hours of germination and reached its maximum value after 48 hours and then declined rapidly. Protein deamidase from germinating wheat seeds was further purified by salting out with phosphate salts and by subsequent chromatography on gel filtration and DEAE-cellulose column chromatography. Polyacrylamide slab gel elec trophoresis showed the purified enzyme to be homogeneous. The molecular weight was determined to be 59 kDa by gel filtration and 60 kDa, by SDS-Polyacrylamide slab gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were found to be 6.9 and 32°C, respectively.The functional properties of wheat gluten were investigated after treating with it purified deamidase enzyme from germinating wheat seeds at pH 6.9 at 32°C. The functional properties such as solubility, emulsification and foam formation of deamidase treated gluten were improved greatly as compared to that of native gluten. The solubility of the deamidase treated gluten was remarkably high in the pH range of 6 to 8, in which the native gluten was insoluble. It was apparent that the improvement of functional properties of wheat gluten was mainly due to the deamidation induced by wheat deamidase at pH 6.9 and 32°C. Key words: Wheat seeds, Deamidase, Deamidation, Wheat gluten, DEAE-cellulose, SDS-PAGE. DOI: http://dx.doi.org/10.3329/jbs.v15i0.2208 J. bio-sci. 15: 89-98, 2007

scholarly journals Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1973 ◽  
Vol 133 (3) ◽  
pp. 541-550 ◽  
Author(s):  
Catherine S. Hawes ◽  
D. J. D. Nicholas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Initial Velocity ◽  
Isotopic Exchange ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzyme Reaction ◽  
Velocity Data ◽  
Sequential Reaction ◽  
Optimum Reaction

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15°C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP2- complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the Km of each substrate was determined at infinite concentration of the other (Km MgATP2-, 0.07mm; MoO42-, 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5′-[35S]sulphatophosphate and SO42-, 35SO42- and adenosine 5′-sulphatophosphate, occurred only in the presence of either MgATP2- or PPi. This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO32-, S2O32- (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO42- was produced by SO42- (Ki=2.0mm) and non-competitive inhibition by sulphide (Ki=3.4mm). 8. Adenosine 5′-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity–substrate curves with both MgATP2- and MoO42- to sigmoidal forms.

Partial purification and properties of a β-glucosidase from Erwinia herbicola Y46

1975 ◽  
Vol 21 (4) ◽  
pp. 513-520 ◽  
Author(s):  
A. Garibaldi ◽  
L. N. Gibbins
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Basal Medium ◽  
Plant Diseases ◽  
Partial Purification ◽  
Major Protein ◽  
Erwinia Herbicola ◽  
Major Protein Band

A constitutive β-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant β-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight." The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-β-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against β-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of β-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells. The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0–7.5 (100% activity) and 4.5–>8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 °C, but only 25% after 30 min at 60 °C. The enzyme had a mean Km value (phloridzin) of 1.35 × 10−4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5–7.0.

Digestive processes of haematophagous insects. XI. Partial purification and some properties of six proteolytic enzymes from the tsetse fly Glossina morsitans morsitans Westwood (Diptera: Glossinidae)

1976 ◽  
Vol 54 (11) ◽  
pp. 1950-1959 ◽  
Author(s):  
R. H. Gooding ◽  
B. M. Rolseth
Keyword(s):  
Proteolytic Enzymes ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Glossina Morsitans Morsitans ◽  
Tsetse Fly ◽  
Partial Purification ◽  
Glossina Morsitans ◽  
Molecular Weights ◽  
Diethylaminoethyl Cellulose

By O-(diethylaminoethyl)cellulose (DEAE-cellulose) chromatography, affinity chromatography, and Sephadex gel filtration, six proteolytic enzymes active in alkaline medium have been found in the digestive portion of the midgut (tissue and lumen contents) of adult Glossina morsitans morsitans. By use of synthetic substrates the enzymes have been characterized as aminopeptidase (AP; EC. 3.4.11.1), carboxypeptidase A (CPA; EC 3.4.12.2), carboxypeptidase B (CPB; EC 3.4.12.3), trypsin (EC 3.4.21.4), a trypsinlike enzyme designated proteinase VI, and a chymotrypsinlike enzyme designated proteinase VII. By Sephadex G-100 gel filtration the molecular weights were estimated to be 20 000 for trypsin, 19 000 for proteinase VI, 35 500 for proteinase VII, 30 000 for CPA, 22 000 for CPB, and [Formula: see text] for AP. The Km values (mg/ml) for haemoglobin were 3.43 for trypsin, 2.45 for proteinase VI, 3.68 for proteinase VII, and 2.42 for CPA. The Km values for casein were 1.22 for trypsin and 1.38 for proteinase VII. Casein showed substrate inhibition when hydrolyzed by proteinase VI and VII. Neither haemoglobin nor casein was hydrolyzed by AP and CPB. The pH optima were determined for hydrolysis of casein and the synthetic substrates.

scholarly journals Regulatory kinetics of wheat-germ aspartate transcarbamoylase. Adaptation of the concerted model to account for complex kinetic effects of uridine 5′-monophosphate

1984 ◽  
Vol 221 (2) ◽  
pp. 281-287 ◽  
Author(s):  
R J Yon
Keyword(s):  
Wheat Germ ◽  
Initial Rate ◽  
Dissociation Constants ◽  
Phosphate Concentration ◽  
Crude Enzyme ◽  
Aspartate Transcarbamoylase ◽  
Phosphate Binding ◽  
Carbamoyl Phosphate ◽  
Rate Versus ◽  
Kinetic Effects

The kinetic effects of the end-product inhibitor UMP on aspartate transcarbamoylase (EC 2.1.3.2) purified to homogeneity from wheat germ were studied. In agreement with an earlier study of the relatively crude enzyme [Yon (1972) Biochem. J. 128, 311-320], the half-saturating concentrations of UMP and of the first substrate, carbamoyl phosphate (but not of the second, L-aspartate), were found to be strongly interdependent. However, the kinetic behaviour of the pure enzyme differed from that of the crude enzyme in several important respects, namely: (a) the apparent affinity for UMP was lower with the pure enzyme; (b) sigmoidicity was absent from plots of initial rate versus carbamoyl phosphate concentration, each at a fixed UMP concentration; (c) sigmoidicity was greatly exaggerated in plots of initial rate versus UMP concentration, each at a fixed carbamoyl phosphate concentration, owing to the occurrence of a slight but definite maximum in each plot at low UMP concentration; (d) there was a relative increase in this maximum in the presence of N-phosphonacetyl-L-aspartate, an inhibitor competitive with carbamoyl phosphate. It is shown that a modified two-conformation concerted-transition model can be used to account for most of these features of the pure enzyme. The model treats carbamoyl phosphate and UMP as antagonistic allosteric ligands binding to alternative conformational states [Monod, Wyman & Changeux (1965) J. Mol. Biol. 12, 88-118], carbamoyl phosphate binding non-exclusively (dissociation constants 20 microM and 85 microM respectively) and UMP binding exclusively (dissociation constant 2.5 microM). The model postulates further that the conformation with lower affinity for carbamoyl phosphate has the higher value of kcat., and that it binds UMP in competition with carbamoyl phosphate. Parameters giving the best fit of experimental data to this model were found by a non-linear least-squares search procedure.

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