Alkaline phosphatases of Neurospora crassa. Part II product inhibition studies

1972 ◽  
Vol 18 (4) ◽  
pp. 407-421 ◽  
Author(s):  
F. W. J. Davis ◽  
Howard Lees
Keyword(s):  
Neurospora Crassa ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Phosphate Hydrolysis ◽  
Non Linear ◽  
Inhibition Studies ◽  
Enzyme Complexes ◽  
Hydrolysis Of

A partially purified preparation of the constitutive alkaline phosphatase from Neurospora crassa, containing two electrophoretically distinct activities was used in initial studies of product inhibition patterns. Inorganic phosphate was shown to be a linear competitive inhibitor, and p-nitrophenol to be a non-linear, non-competitive inhibitor of p-nitrophenyl phosphate hydrolysis. Glycerol was shown to be a linear non-competitive inhibitor of β-glycerophosphate hydrolysis.A purification procedure whereby one enzyme activity could be obtained free of the second was devised. The purified enzyme catalyzed the hydrolysis of a wide range of substrates and had a molecular weight of 111 000. Its hydrolysis of glucose 6-phosphate was competitively inhibited by phosphate and non-competitively inhibited by glucose. Both inhibitions were linear. Hydrolysis of p-nitrophenyl phosphate was competitively inhibited by phosphate in a linear manner, but p-nitrophenol was a non-linear, non-competitive inhibitor. Alternate product inhibition by glucose was linear competitive. No inhibition by p-nitrophenol of glucose 6-phosphate hydrolysis could be detected.The inhibition data for glucose 6-phosphate and β-glycerophosphate may be consistent with an ordered Uni-Bi mechanism expanded to include one or more isomerizations of enzyme complexes. The postulation of a different mechanism involving alternate pathways is probably required to explain the data obtained when p-nitrophenyl phosphate was the substrate.

scholarly journals Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

1972 ◽  
Vol 127 (1) ◽  
pp. 87-96 ◽  
Author(s):  
P. G. Bolton ◽  
A. C. R. Dean
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Continuous Culture ◽  
Activity Profile ◽  
Glucose Effect ◽  
Ph Values ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (Km 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO43−-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F−, PO43−) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.

Monoacylglycerol lipase activity in cardiac myocytes

1988 ◽  
Vol 66 (9) ◽  
pp. 1013-1018 ◽  
Author(s):  
David L. Severson ◽  
Mariette Hee-Cheong
Keyword(s):  
Lipase Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Fatty Acyl ◽  
Supernatant Fraction ◽  
Monoacylglycerol Lipase ◽  
Pellet Fraction ◽  
Rat Hearts ◽  
Triton X ◽  
Hydrolysis Of

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100 000 × g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100 000 × g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 μM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 μM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA > free fatty acids > fatty acyl carnitine.

scholarly journals Artemia purine phosphoribosyltransferases. Purification and characterization

1991 ◽  
Vol 275 (2) ◽  
pp. 327-334 ◽  
Author(s):  
C Montero ◽  
P Llorente
Keyword(s):  
Divalent Cations ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Ph Profile ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Artemia Cysts ◽  
Inhibition Studies

Adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) have been purified from Artemia cysts and nauplii to apparent homogeneity, as determined by SDS-PAGE. The purification includes affinity chromatography on AMP-Sepharose, which binds both enzymes, and they are eluted at different 5-phospho-alpha-D-ribosyl diphosphate (PP-Rib-P) concentrations. The purified enzymes from Artemia cysts were similar to nauplii enzymes with respect to Mr in denaturing gel electrophoresis and gel filtration, pH and cation dependence and kinetic constants for substrates and inhibitors. By Sephadex G-100 filtration, the native Mr of the adenine and hypoxanthine-guanine enzymes was estimated to be Mr 28,000 and 66,000, respectively. Analysis by SDS-PAGE revealed that the APRTase was a dimer of Mr 15,000 sub-units and the HGPRTase, a tetramer of four identical Mr 19,000 sub-units. The pH profile of the HGPRTase shows two apparent buffer-independent pH optima, at 7.0 and 9.5, while the APRTase has just one, at about pH 8-9. The purine phosphoribosyltransferase activity with adenine was highest, about tenfold the HGPRTase activity with hypoxanthine and fivefold that with guanine. Both enzymes exhibited similar requirements for divalent cations, either Mg2+, Mn2+ or Zn2+, while Ca2+ is highly inhibitory. The Km values of APRTase for adenine and PP-Rib-P are 2 and 30 microM, respectively, and the Km values of HGPRTase for hypoxanthine, guanine and PP-Rib-P are less than 1, less than 1 and 15 microM, respectively. Plots of the reciprocal enzyme activities versus reciprocal concentrations of one substrate at several fixed levels of the second one yield a pattern of inhibition by guanine and hypoxanthine. Product-inhibition studies indicated that AMP is a competitive inhibitor with respect to PP-Rib-P in the APRTase reaction, while the HGPRTase shows a mixed inhibition by GMP.

scholarly journals Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 833-846 ◽  
Author(s):  
M C Scrutton ◽  
I Beis
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Detectable Effect ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

Alkaline phosphatases of Neurospora crassa. Part III. Effects of pH and mechanism of action

1973 ◽  
Vol 19 (1) ◽  
pp. 135-146 ◽  
Author(s):  
F. W. J. Davis ◽  
Howard Lees
Keyword(s):  
Neurospora Crassa ◽  
Substrate Concentration ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Ph Effects ◽  
Alkaline Phosphatases ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
Hydroxyl Ion ◽  
Effects Of Ph

The effects of pH on the activity of a highly purified constitutive alkaline phosphatase from Neurospora crassa were studied. The pH optimum for the reaction was a linear function of the logarithm of the substrate concentration, as is typical for alkaline phosphatases. Other pH effects were complex and Vmax or pKm versus pH plots differed for the two substrates used and from similar plots prepared with the repressible enzyme from this or other sources. The Km for p-nitrophenyl phosphate increased 200-fold over the pH range 7.0–8.7. Substrate inhibition was more pronounced at low than high pH. Lineweaver-Burk plots were non-linear at high substrate concentration but linear at low substrate concentration. Substrate inhibition by glucose 6-phosphate was not observed.The data are consistent with the hypothesis that hydroxyl ion is a second substrate for the enzyme and is an activator at high and an inhibitor at low p-nitrophenyl phosphate and glucose 6-phosphate concentrations. A Bi-Bi mechanism with random substrate addition, dead-end substrate, and product inhibition, having ordered release of products is discussed and is consistent with kinetic and pH studies. A two-site model for the active center of the enzyme is presented to account for alternate product inhibition data.

Acid phosphatase activity demonstrated by intact Angiostrongylus cantonensis with special reference to its function

Parasitology ◽  
1979 ◽  
Vol 79 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Jun Maki ◽  
Toshio Yanagisawa
Keyword(s):  
Acid Phosphatase ◽  
External Medium ◽  
Reciprocal Inhibition ◽  
Angiostrongylus Cantonensis ◽  
Phosphate Esters ◽  
Glucose Phosphate ◽  
Nitrophenyl Phosphate ◽  
Inhibition Studies ◽  
Hydrolysis Of

SUMMARYIntact Angiostrongylus cantonensis is able to hydrolyse glucose-phosphate esters, mononucleotides and p-nitrophenyl phosphate as well as β-glycerophosphate in vitro. Reciprocal inhibition studies suggest that the hydrolysis of such substrates is due to a non-specific phosphomonoesterase. Molybdate ions, which exert no effect on either the uptake of glucose or the production of lactate, inhibit the hydrolysis of glucose-1- phosphate in the external medium and simultaneously lower the production of lactate by the intact worms in vitro.

Influence of pH on the Kinetics of Reactions Catalyzed by Alkaline Phosphatase

1973 ◽  
Vol 51 (7) ◽  
pp. 1096-1103 ◽  
Author(s):  
Irwin Hinberg ◽  
Keith J. Laidler
Keyword(s):  
Alkaline Phosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Coordinated Water ◽  
Ph Range ◽  
Barbital Buffer ◽  
Influence Of Ph ◽  
Kinetics Of ◽  
Hydrolysis Of

An experimental study has been made of the kinetics of the hydrolysis of p-nitrophenyl phosphate catalyzed by chicken-intestinal alkaline phosphatase. The work was done in barbital buffer (carbonate above pH 9.6), and covered the pH range from 7.0 to 10.0. A sufficiently wide range of substrate concentration was used to allow reliable values of [Formula: see text] and [Formula: see text] to be determined. The results lead to pK values of 8.1 and 8.6 for the free enzyme, and it is concluded that the Michaelis complex and the phosphoryl intermediate ionize only on the acid side, the former also having a pK of 8.1. It is suggested that the group of pK 8.1 is probably an α-amino group and that the group of pK 8.6 probably corresponds to the ionization of a Zn(II)-coordinated water molecule.

Inositol phosphatase activity of theEscherichia coli agp-encoded acid glucose-1-phosphatase

2002 ◽  
Vol 48 (9) ◽  
pp. 801-809 ◽  
Author(s):  
Michael A Cottrill ◽  
Serguei P Golovan ◽  
John P Phillips ◽  
Cecil W Forsberg
Keyword(s):  
Escherichia Coli ◽  
Hplc Analysis ◽  
Inositol Phosphate ◽  
Gene Library ◽  
Inositol Phosphatase ◽  
Ph Values ◽  
Hydrolysis Products ◽  
Nitrophenyl Phosphate ◽  
Phosphate Hydrolysis ◽  
Hydrolysis Of

When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55°C, while InsP6and p-nitrophenyl phosphate were hydrolyzed maximally at 60°C. The Agp exhibited Kmvalues of 0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5, D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase. Key words: phosphatase, phytate, bacteria, inositol phosphate, phytase.

Enzyme-assisted Extraction for Optimized Recovery of Phenolic Bioactives from Peganum hermala Leaves Using Response Surface Methodology

2018 ◽  
Vol 17 (4) ◽  
pp. 349-354
Author(s):  
Qadir Rahman ◽  
Anwar Farooq ◽  
Amjad Gilani Mazhar ◽  
Nadeem Yaqoob Muhammad ◽  
Ahmad Mukhtar
Keyword(s):  
Response Surface Methodology ◽  
Response Surface ◽  
Enzyme Concentration ◽  
Coumaric Acid ◽  
Assisted Extraction ◽  
Pre Treatment ◽  
Optimized Conditions ◽  
Enzyme Complexes ◽  
Hydrolysis Of ◽  
Central Composite

This study investigates the effect of enzyme formulations (Zympex-014, Kemzyme dry-plus and Natuzyme) on recovery of phenolics from Peganum hermala (harmal) leaves, under optimized conditions using response surface methodology. As compared to the other enzyme complexes, the yield (34 g/100g) obtained through Zympex-014-assisted extraction was higher under optimized conditions such as time (75 min), temperature (70°C), pH (6.5) and enzyme concentration (5 g/100 g) using central composite design (CCD). Effectiveness of Zympex-014 towards hydrolysis of P. hermala leaves cell wall was examined by analyzing the control and enzyme-treated leave residues using scanning electron microscope (SEM). GC/MS characterization authenticated the presence of quercetin (1.44), gallic acid (0.23), caffeic acid (0.04), cinnamic acid (0.05), m-coumaric acid (0.23) and p-coumaric acid (0.37 μg/g) as the potent phenolics in Zympex-014 based extract. It can be concluded from the findings of the current work that pre-treatment of P. hermala leaves with Zympex-014 significantly enhanced the recovery of phenolics that supports its potential uses in the nutra-pharamaceutical industry.

scholarly journals Renal sodium-potassium adenosine triphosphatase. Optical localization and x-ray microanalysis.

1975 ◽  
Vol 23 (11) ◽  
pp. 828-839 ◽  
Author(s):  
R Beeuwkes ◽  
S Rosen
Keyword(s):  
Atpase Activity ◽  
Electron Probe ◽  
Sequential Analysis ◽  
Relative Sensitivity ◽  
Initial Reaction ◽  
Initial Product ◽  
Sodium Potassium ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of ◽  
Convoluted Tubules

The distribution of sodium-potassium adenosine triposphatase (Na-K-ATPase) activity in kidney sections has been studied by a method based on the hydrolysis of p-nitrophenyl phosphate in alkaline medium containing dimethyl sulfoxide. The products at each stage in the reaction sequence have been subjected to electron probe microanalysis. The initial product was identified as a mixture of KMgPO4 and Mg(PO4)2, and sequential analysis demonstrated the linearity of conversion of this product to a visible form. In human, rabbit and rat kidneys the distribution of activity was found to be essentially identical, with highest levels located in thick ascending limbs and distal convoluted tubules. The initial reaction was completely potassium dependent and was inhibited by ouabain in concentrations reflecting the relative sensitivity of microsomal Na-K-ATPase in each species. Measurement of initial product phosphorus by means of the electron probe is presented as a practical technique for direct quantitation of Na-K-ATPase activity in identified tubule segments.

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