THE WHEAT LEAF PHOSPHATASES: VII. FURTHER STUDIES ON INHIBITORS AT pH 5.7

A survey of the inhibitory effect of various ribonucleosides, deoxyribonucleosides, sugars, phenols, and vitamins on the hydrolysis of 17 phosphatase substrates has been made. The more soluble ribonucleosides and deoxyribonucleosides inhibited the enzymatic hydrolysis of adenosine 5′-phosphate, phenolphthalein diphosphate, phenylphosphate, p-nitrophenyl phosphate, and in some cases the hydrolysis of adenosine 3′-phosphate, adenosine 2′-phosphate, and riboflavin 5′-phosphate. A 0.02 M concentration of orthophosphate inhibited the hydrolysis of all the compounds tested except adenosine 5′-phosphate and phenolphthalein diphosphate.These results, together with earlier findings, are discussed in terms of the concept that wheat leaf press juice contains two types of acid phosphatase, namely, β-glycerophosphatase and adenosine 5′-phosphatase. These two types of enzyme appear to have partially overlapping substrate specificities.

Download Full-text

Evidence for the regulation of guinea-pig heart microsomal phosphatidylcholine-hydrolysing phospholipase A1 by guanosine 5′-[γ-thio]triphosphate

Biochemical Journal ◽

10.1042/bj2880965 ◽

1992 ◽

Vol 288 (3) ◽

pp. 965-968 ◽

Cited By ~ 3

Author(s):

K Badiani ◽

X Lu ◽

G Arthur

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Guinea Pig ◽

Molecular Species ◽

Inhibitory Effect ◽

Phospholipase A1 ◽

Guinea Pig Heart ◽

Substrate Specificities ◽

Rate Of Hydrolysis ◽

Hydrolysis Of

We have recently characterized lysophospholipase A2 activities in guinea-pig heart microsomes and postulated that these enzymes act sequentially with phospholipases A1 to release fatty acids selectively from phosphatidylcholine (PC) and phosphatidylethanolamine, thus providing an alternative route to the phospholipase A2 mode of release. In a further investigation of the postulated pathway, we have characterized the PC-hydrolysing phospholipase A1 in guinea-pig heart microsomes. Our results show that the enzyme may have a preference for substrates with C16:0 over C18:0 at the sn-1 position. In addition, although the enzyme cleaves the sn-1 fatty acid, the rate of hydrolysis of PC substrates with C16:0 at the sn-1 position was influenced by the nature of the fatty acid at the sn-2 position. The order of decreasing preference was C18:2 > C20:4 = C18:1 > C16:0. The hydrolyses of the molecular species were differentially affected by heating at 60 degrees C. An investigation into the effect of nucleotides on the activity of the enzyme showed that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) inhibited the hydrolysis of PC by phospholipase A1 activity, whereas GTP, guanosine 5′-[beta-thio]diphosphate (GDP[S]), GDP, ATP and adenosine 5′-[gamma-thio]triphosphate (ATP[S]) did not affect the activity. The inhibitory effect of GTP[S] on phospholipase A1 activity was blocked by preincubation with GDP[S]. A differential effect of GTP[S] on the hydrolysis of different molecular species was also observed. Taken together, the results of this study suggest the presence of more than one phospholipase A1 in the microsomes with different substrate specificities, which act sequentially with lysophospholipase A2 to release linoleic or arachidonic acid selectively from PC under resting conditions. Upon stimulation and activation of the G-protein, the release of fatty acids would be inhibited.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj1270087 ◽

1972 ◽

Vol 127 (1) ◽

pp. 87-96 ◽

Cited By ~ 28

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.

Download Full-text

Polyphosphate body and acid phosphatase localization in Nostoc sp.

Canadian Journal of Microbiology ◽

10.1139/m84-002 ◽

1984 ◽

Vol 30 (1) ◽

pp. 8-15 ◽

Cited By ~ 3

Author(s):

John D. DuBois ◽

Keith R. Roberts ◽

Lawrence A. Kapustka

Keyword(s):

Enzyme Activity ◽

Acid Phosphatase ◽

Phosphatase Activity ◽

Acid Phosphatase Activity ◽

Energy Stress ◽

Nitrophenyl Phosphate ◽

Carbon Compounds ◽

Electron And Light Microscopy ◽

Polyphosphate Bodies ◽

Hydrolysis Of

Polyphosphate bodies and acid phosphatase activity were characterized in Nostoc sp. to determine if the hydrolysis of polyphosphate bodies occurs during dark (energy stress) periods. Electron and light microscopy were used to locate polyphosphate bodies. Acid phosphatase activity was measured using p-nitrophenyl phosphate as the substrate to determine net changes in the level of the enzyme activity. To induce energy stress, Nostoc sp. cells were kept in the dark for 72 h to deplete stored carbon compounds. Cells incubated in the light for 72 h (controls) showed acid phosphatase activity localized around the perimeter of polyphosphate bodies. When cells were incubated in the dark, acid phosphatase activity occurred throughout the polyphosphate body matrix. However, complete hydrolysis of the polyphosphate body did not occur and the rate of acid phosphatase activity was not affected.

Download Full-text

Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic β-cells

Biochemical Journal ◽

10.1042/bj1620009 ◽

1977 ◽

Vol 162 (1) ◽

pp. 9-18 ◽

Cited By ~ 22

Author(s):

L Å Idahl ◽

Å Lernmark ◽

J Sehlin ◽

I B Täljedal

Keyword(s):

Plasma Membranes ◽

Islet Cells ◽

Protective Action ◽

Inhibitory Effect ◽

Dependent Manner ◽

Nitrophenyl Phosphate ◽

Cation Pump ◽

Ion Pumping ◽

Hydrolysis Of

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.

Download Full-text

The enzymatic method for the quantitative determination of benzalkonium chloride in the antiseptic solution “CUTASEPT® F”

Žurnal organìčnoï ta farmacevtičnoï hìmìï ◽

10.24959/ophcj.21.244359 ◽

2021 ◽

Vol 19 (4(76)) ◽

pp. 33-39

Author(s):

Olena V. Koval’ska ◽

Mykola Ye. Blazheyevskіy

Keyword(s):

Enzymatic Hydrolysis ◽

Quantitative Determination ◽

Peracetic Acid ◽

Benzalkonium Chloride ◽

Chloride Content ◽

Inhibitory Effect ◽

Enzymatic Method ◽

Limit Of Quantitation ◽

Hydrolysis Of

Aim. To develop an alternative method for the quantitative determination of the benzalkonium chloride content as an active pharmaceutical ingredient in the disinfectant solution “CUTASEPT® F”.Materials and methods. The method is based on the ability of benzalkonium chloride to inhibit the enzymatic hydrolysis of acetylcholine by acetylcholinesterase. The reaction rate is assessed by the non-hydrolyzed acetylcholine residue, which is determined by the amount of peracetic acid produced during the interaction with the excess of the hydrogen peroxide solution. The indicator reaction is the interaction of p-phenetidine with peracetic acid that leads to the formation of 4,4’-azoxyphenetole with λmax = 358 nm (log10 ε = 4.2).Results and discussion. As a result of the research conducted the linear dependence of the degree of inhibition of the enzymatic hydrolysis of acetylcholine (U, %) on the concentration of benzalkonium chloride was determined in the concentration range of (0.5 – 7.0) × 10–6 mol L-1 with the correlation coefficient of 0.999. The limit of quantitation was 1.9 × 10–6 mol L-1.Conclusions. As a result of the research conducted the kinetic enzymatic method for the quantitative determination of benzalkonium chloride has been developed by its inhibitory effect in the biochemical reaction of acetylcholine hydrolysis. This method is fast, cheap and easy to perform, does not require expensive equipment, and available for use in the field.

Download Full-text

The inhibitory effect of xylan on enzymatic hydrolysis of cellulose is dependent on cellulose ultrastructure

Cellulose ◽

10.1007/s10570-020-03087-9 ◽

2020 ◽

Vol 27 (8) ◽

pp. 4417-4428 ◽

Cited By ~ 1

Author(s):

Xindong Chen ◽

Lian Xiong ◽

Hailong Li ◽

Liquan Zhang ◽

Ge Yuan ◽

...

Keyword(s):

Enzymatic Hydrolysis ◽

Inhibitory Effect ◽

Enzymatic Hydrolysis Of Cellulose ◽

Hydrolysis Of Cellulose ◽

Hydrolysis Of

Download Full-text

Isolation and partial characterization of an acid phosphatase from Artemisia vulgaris pollen extract

Journal of the Serbian Chemical Society ◽

10.2298/jsc0209567c ◽

2002 ◽

Vol 67 (8-9) ◽

pp. 567-572 ◽

Cited By ~ 4

Author(s):

Tanja Cirkovic-Velickovic ◽

Marija Gavrovic-Jankulovic ◽

Mirjana Bukilica ◽

Ljuba Mandic ◽

Spomenka Petrovic ◽

...

Keyword(s):

Acid Phosphatase ◽

Gel Filtration ◽

Sulfhydryl Group ◽

Inhibitory Effect ◽

Tween 20 ◽

Pollen Extract ◽

Gel Chromatography ◽

Artemisia Vulgaris ◽

Nitrophenyl Phosphate

An acid phosphatase from an extract of mugwort (Artemisia vulgaris) pollen was purified by a factor of 48 by a combination of ion exchange and gel-chromatography. The molecular weights of the enzyme were 76 kDa and 73 kDa, determined by gel filtration on a Sephadex G-100 sf column and by SDS PAGE(under reducing and non-reducing conditions), respectively. In analytical isoelectrofocusing, the enzyme appears as two very close bands pI at about 4.2. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.16mM. The purified enzyme has broad specificity, and hydrolyses p-nitrophenyl phosphate and ?-naphthyl phosphate. Pyrophosphate and O-phospho-L-tyrosine were estimated to be the best substrates for this enzyme as potential in vivo substrates. The enzyme is inhibited competitively by phosphate (Ki = 1.25 mM), molybdate (Ki = 0.055 mM) and pyrophosphate (Ki = 6.7 mM) and non-competitively by fluoride (Ki = 9.8 mM). Metal ions such as Hg2+, Cu2+ and Zn2+ express an inhibitory effect on the enzyme, while the enzyme is slightly activated by non-ionic detergents, Tween 20 and Triton X-100. There is no change in the enzyme activity in the presence of tartrate, citrate, EDTA, 1,10-phenanthroline and sulfhydryl-group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.

Download Full-text

Improvement of the method of obtaining human IgA Fc-fragments

Visnyk of Dnipropetrovsk University Biology medicine ◽

10.15421/021506 ◽

2015 ◽

Vol 6 (1) ◽

pp. 32-35

Author(s):

O. Y. Galkin ◽

Y. V. Gorshunov ◽

V. F. Solovjova

Keyword(s):

Enzymatic Hydrolysis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Inhibitory Effect ◽

Nitrogen Atmosphere ◽

Maximum Output ◽

Serological Tests ◽

Isolation And Purification ◽

Antibody Conjugate ◽

Hydrolysis Of

To address a number of fundamental and applied problems in immunology, molecular and cellular biology and biotechnology it is necessary to obtain Fc-fragments of immunoglobulins. Fc-fragments may be used for studying of the effector functions of antibodies which are mediated by these areas. They are often used as an immunogen to produce anti-specie (based on so-called secondary antibody) conjugate in the development of serological tests for diagnostics (predominantly such conjugate based on monoclonal antibodies). The work is aimed to develop improved methods of obtaining and allocation of Fc-fragments of human IgA. To achieve this objective, optimization of hydrolysis of IgA with subsequent purification of Fс-fragments have been carried out. Improved method of obtaining Fc-fragments of IgA provides: papain hydrolysis of immunoglobulin in the environment of nitrogen for 4 h, allowing to achieve maximum output of Fc-fragments without their further degradation: isolation and purification of Fc-fragments of human IgA by one-stage gel filtration on sephadex G-100; control of purity of the target product by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and Ouchterlony immunodiffusion. Enzymatic hydrolysis was carried out at the optimal temperature of papain (37 °C). As the oxygen in the air may have inhibitory effect on enzymatic hydrolysis reaction, the reaction mixture was incubated in the nitrogen atmosphere to prevent inactivation of papain. To reduce the incident degradation of immunoglobulin molecules, papain hydrolysis was carried out without using an enzyme activator (cysteine). Usage of the proposed scheme allows obtaining Fc-fragments of human IgA of high purity. Outcome of Fc-fragments after all stages of purification was about 18% of the initial amount of IgA in the preparation. Molecular weight from Fc-fragments of human IgA was equal to approximately 70 kDa.

Download Full-text

THE NON-ENZYMATIC HYDROLYSIS OF p-NITROPHENYL PHOSPHATE

Canadian Journal of Chemistry ◽

10.1139/v58-096 ◽

1958 ◽

Vol 36 (4) ◽

pp. 686-690 ◽

Cited By ~ 11

Author(s):

K. A. Holbrook ◽

Ludovic Ouellet

Keyword(s):

Activation Energy ◽

Enzymatic Hydrolysis ◽

Ionic Species ◽

Kcal Mole ◽

First Order ◽

Colorimetric Measurement ◽

Nitrophenyl Phosphate ◽

Ph Range ◽

Kinetics Of ◽

Hydrolysis Of

The kinetics of the non-enzymatic hydrolysis of p-nitrophenyl phosphate have been studied in aqueous solution in the pH range 2.6 to 9.0 and at temperatures from 68.0°to 82.0 °C. The reaction has been followed by colorimetric measurement of the nitrophenol produced by the reaction[Formula: see text]The reaction is first order with respect to p-nitrophenyl phosphate and has an activation energy of 26.0 kcal./mole at pH 2.6. An explanation has been proposed in terms of the different rates of hydrolysis of the various ionic species of the ester present in solution.

Download Full-text