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.

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.

scholarly journals Acid phosphatase of the yeast Saccharomyces cerevisiae. Purification and properties of the enzyme

2015 ◽  
Vol 46 (2) ◽  
pp. 173-186 ◽  
Author(s):  
W. Wątorek ◽  
B. Morawiecka
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acid Phosphatase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fluoride Ions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purification And Properties ◽  
Total Molecular Weight ◽  
C 50 ◽  
Sepharose 4B

Acid phosphatase from the yeast <i>Saccharomyces cerevisiae</i> was purified to homogeneity as ascertained by ultracentrifugation and electrophoresis. The purification procedure involved mechanical cell disruption, ethanol precipitation, chromatography on DEAE-cellulose, gel filtration on Sepharose 4B. The sedimentation constant S20<sup>0.580</sup> of the purified enzyme was 15.4 S. Carbohydrate content accounted for 50% of the total molecular weight of the enzyme. The optimum pH for purified enzyme was 3.0-3.5, it was stable at pH 3.0-5.0 at room temperature. After 10 min. incubation at 45° C, 50 per cent of the enzymatic activity was lost. Michaelis constant was found to be 1.3 x 10<sup>-4</sup> M for p-nitrophenylphosphate and 5 x 10<sup>-4</sup> M for 3-glycerophosphate as substrates. The enzyme was inhibited by Hg<sup>2+</sup>, Cu<sup>2+</sup>, Fe<sup>3+</sup>, molybdate, phosphate, arsenate, fluoride ions. Inhibition caused by fluoride ions was noncompetitive, by phosphate - competitive, 5 M urea inactivated the enzyme completely, inactivation was reversible at urea concentration below 2,5 M.

scholarly journals Kinetic Analysis of Guanidine Hydrochloride Inactivation of β-Galactosidase in the Presence of Galactose

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Charles O. Nwamba ◽  
Ferdinand C. Chilaka
Keyword(s):  
Rate Constants ◽  
Initial Velocity ◽  
Competitive Inhibition ◽  
Guanidine Hydrochloride ◽  
Free Enzyme ◽  
Velocity Data ◽  
Substrate Complex ◽  
Inactivation Rate Constant ◽  
Enzyme Substrate ◽  
Straight Lines

Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant Km and Vmax values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([P]∞−[P]t) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[P]∞ versus 1/[S] yielded k+0 and k'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k+0 and k'+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed.

Anticoagulants Produced by Thrombin from Fibrin, the Effect on Blood Coagulation, Some Physical Characteristics

1971 ◽  
Vol 26 (02) ◽  
pp. 211-223 ◽  
Author(s):  
Ch R. Muirhead ◽  
D. C Triantaphyllopoulos
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fibrin Clot ◽  
Disc Electrophoresis ◽  
Physical Characteristics ◽  
Advanced Stage ◽  
Kallikrein Inhibitor ◽  
Shed Blood ◽  
Complete Lysis

SummaryChromatographed thrombin in the presence of both 50 Kallikrein inhibitor units of Trasylol per ml and 0.1 M E-ACA solubilized fibrin and the products of lysis possessed anticoagulant properties. The peak of the antithrombic activity coincided with the time of complete lysis of the fibrin clot, plasmin lysed fibrin exhibited the peak of its antithrombic activity much earlier. The effect of thrombin lysed fibrin on the prothrombin consumption of shed blood was found to be inhibitory.The products of the digestion of fibrin by thrombin and by plasmin, isolated at an advanced stage of proteolysis were compared by gel filtration, disc electrophoresis and DEAE cellulose chromatography. Differences in physical characteristics of these fibrin breakdown products offer evidence that they were produced by two different enzymes.

Inhibition of Yeast Growth by Tryptamine and Recovery with Tryptophan

2020 ◽  
Vol 16 (1) ◽  
pp. 48-52 ◽  
Author(s):  
Chandrika Kadkol ◽  
Ian Macreadie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Competitive Inhibition ◽  
Yeast Species ◽  
Inhibitory Effect ◽  
The Body ◽  
Inhibitory Effects ◽  
Trna Synthetases ◽  
Fermentative Growth ◽  
Biogenic Monoamine

Background: Tryptamine, a biogenic monoamine that is present in trace levels in the mammalian central nervous system, has probable roles as a neurotransmitter and/or a neuromodulator and may be associated with various neuropsychiatric disorders. One of the ways tryptamine may affect the body is by the competitive inhibition of the attachment of tryptophan to tryptophanyl tRNA synthetases. Methods: This study has explored the effects of tryptamine on growth of six yeast species (Saccharomyces cerevisiae, Candida glabrata, C. krusei, C. dubliniensis, C. tropicalis and C. lusitaniae) in media with glucose or ethanol as the carbon source, as well as recovery of growth inhibition by the addition of tryptophan. Results: Tryptamine was found to have an inhibitory effect on respiratory growth of all yeast species when grown with ethanol as the carbon source. Tryptamine also inhibited fermentative growth of Saccharomyces cerevisiae, C. krusei and C. tropicalis with glucose as the carbon source. In most cases the inhibitory effects were reduced by added tryptophan. Conclusion: The results obtained in this study are consistent with tryptamine competing with tryptophan to bind mitochondrial and cytoplasmic tryptophanyl tRNA synthetases in yeast: effects on mitochondrial and cytoplasmic protein synthesis can be studied as a function of growth with glucose or ethanol as a carbon source. Of the yeast species tested, there is variation in the sensitivity to tryptamine and the rescue by tryptophan. The current study suggests appropriate yeast strains and approaches for further studies.

Kinetic Behaviour of Amylase According to pH: A New Perspective for Starch Hydrolysis Process

2020 ◽  
Vol 16 (2) ◽  
pp. 135-144
Author(s):  
Ravneet K. Grewal ◽  
Baldeep Kaur ◽  
Gagandeep Kaur
Keyword(s):  
Metal Ions ◽  
Competitive Inhibition ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Cost Effective ◽  
Starch Hydrolysis ◽  
Divalent Metal Ions ◽  
Activity Data ◽  
Time Saving ◽  
Alkaline Conditions

Background: Amylases are the most widely used biocatalysts in starch saccharification and detergent industries. However, commercially available amylases have few limitations viz. limited activity at low or high pH and Ca2+ dependency. Objective: The quest for exploiting amylase for diverse applications to improve the industrial processes in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence of metal ions as a function of pH. Methods: The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of ions, as a function of substrate concentration in Lineweaver-Burk Plot. Results: Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and 7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5. Conclusion: These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore, a time-saving and cost-effective solution is proposed to overcome the challenges of the existing methodology of starch hydrolysis in starch and detergent industries.

INHIBITION BY HUMAN SERUM OF THE ADIPOKINETIC EFFECT OF A HUMAN PITUITARY LIPID-MOBILIZING FACTOR

1972 ◽  
Vol 71 (3) ◽  
pp. 443-453 ◽  
Author(s):  
Olav Trygstad ◽  
Irene Foss
Keyword(s):  
Human Serum ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Disc Electrophoresis ◽  
Inhibitory Protein ◽  
Human Pituitary ◽  
Albumin Fraction ◽  
Pituitary Glands ◽  
Normal Human

ABSTRACT A lipid-mobilizing factor (LMF) with an adipotrophic effect in human and animal fat tissue has been prepared from human pituitary glands. The addition of normal human serum to LMF reduced its lipolytic effect, and it was completely abolished by serum from a group of obese patients, whereas the lipolysis was not influenced by serum from patients with generalized lipodystrophy. By DEAE-cellulose chromatography of human serum the inhibitory effect on LMF was found to be present in a protein fraction less acidic than the main serum albumin fraction. The inhibitory fraction was deprived of some contaminants by Sephadex gel filtration. Disc electrophoresis demonstrated the presence of three components in the inhibitory protein (IP), and they were identified as albumin, transferin, and haemopexin by immuno-electrophoresis. Precipitation of these proteins by their rabbit antisera demonstrated that the inhibitory effect was present in the albumin fraction. Insulin like activity was not observed in IP. A protein binding of LMF by IP could not be demonstrated. Incubation at 37°C for one hour of a mixture of LMF and IP eliminated the electrophoretic picture of LMF. It is concluded that the inhibitory effect of human serum may be due to proteolysis of LMF.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

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