Preparation Of Phosphatase Research Articles

Purification and Properties of a Nonspecific Acid Phosphatase from Wheat Germ

While studying the phosphoglyceric acid mutase of wheat germ, we noted marked phosphatase activity when extensively purified mutase preparations were tested with n-3-phosphoglyceric acid and D-Z, 3diphosphoglyceric acid as substrates (1). Since we had found low phosphatase activity for these reagents in many biological materials (2), there was the possibility, previously discussed (l), that the enzymatic hydrolysis of these substrates was a dual function of the purified wheat germ mutase. Further examination, however, showed there was no correlation between phosphatase and mutase activities at several stages of purification, indicating phosphatase contamination of the wheat germ mutase preparations. Additional evidence for the nonidentity of the two enzymatic activities has now been obtained by extensive purification of the phosphatase from wheat germ, by the method presented in this paper. The purified preparations of nonspecific acid phosphatase described in this paper have activities of the same order of magnitude as those of the extensively purified intestinal monoesterase (3) and of the crystalline pyrophosphatase of yeast (4). The best phosphatase preparations are essentially free of P-glycerate mutase activity.

The hydrolysis of glucose monophosphates by a phosphatase preparation from pea seeds.

The hydrolysis of glucose monophosphates by a phosphatase preparation from pea seeds.

Histochemical effects of x-radiation.

BS>Histochemical preparations of alkaline and acid phosphatase, esterase, and 5-nucleotidase have demonstrated that doses of x radiation varying from 150 to 800 r affect the activity of these enzimes in liver, spleen, and adrenal but not in kidney. Alkaline phosphatase and 5-nucleotidase were most affected by the radiation and acid phosphatase and esterase least affected. Succinic dehydrogenase activity was markedly reduced in the liver after irradiation by 600 r. (auth)

Action of Purified Milk Phosphatase on Phosphoserine and on Casein

Summary The action of a purified alkaline phosphatase from milk on casein and phosphoserine has been studied. The milk phosphatase preparation has no proteolytic action on casein, but it splits inorganic phosphate from both casein and phosphoserine. The action of the milk phosphatase was studied at pH 8 to 9.5 with several concentrations of phosphoserine. The optimum action was at pH 9.5 or higher. Values for the kinetic constants KM and V max. were calculated from the data. The action of the milk phosphatase on phosphoserine was similar to its action on other simple phosphate esters. The milk phosphatase was considerably less reactive with casein (about 200 times more phosphatase was required for comparable release of phosphate), and the greatest activity was at pH 6 to 7. Reasons are given for not necessarily postulating a second enzyme for this activity, but rather that it might result from the complex nature and size of the casein molecule. Both calcium-sensitive α-casein and whole casein were dephosphorylated at comparable rates. The experiments were carried to 80% dephosphorylation of the casein, and presumably complete dephosphorylation could be accomplished with this phosphatase preparation. It is suggested that some dephosphorylation of casein might occur in milk and that special precautions may be required to obtain casein preparations of maximum and constant phosphorus content.

The co-enzyme factor of alkaline phosphatase

1. 1. The alkaline phosphatases of kidney, intestine and placenta have been extracted by autolysis and butanol procedures. Autolysis yielded more potent preparations with kidney, butanol with intestine and placenta. 2. 2. Electrodialysis resulted in loss of activity by all preparations, which was fully or partially restored by Mg ++. 3. 3. Electrodialysis of the kidney phosphatase, prepared by autolysis, yielded a cathode-chamber fluid which partially reactivated, and with Mg ++ fully reactivated, the de-activated enzyme. No activating property was found in the cathode fluid (or anode) of any of the butanol-extracted phosphatase preparations, or in those of intestine and placenta by autolysis. Kidney phosphatase, prepared by autolysis, may be more dissociable than other preparations. 4. 4. Co ++ like Mg ++ was activating to kidney phosphatase (prepared by both autolysis and butanol extraction) but not to the intestinal and placental preparations. Zn was inhibiting. Uridylic acid activated kidney and intestine autolyzed preparations, and inhibited the others. The effect of amino acids was variable. 5. 5. Chromatographic behaviour, with different solvents, was uniform for placental preparations, variable for kidney and intestine. Electrophoresis showed the enzyme activity of all preparations to be in the α 2-globulin region. Differences in electrophoretic pattern appeared to be due mainly to accompanying inactive protein.

Isolation of 32P-labeled Phosphorylserine from a Purified Preparation of Alkaline Bone Phosphatase Incubated with Radioactive Inorganic Phosphate.

Isolation of 32P-labeled Phosphorylserine from a Purified Preparation of Alkaline Bone Phosphatase Incubated with Radioactive Inorganic Phosphate.

Experiments on electrophoresis in segmental systems composed of polyurethane foam

The use of foamed polyurethane sponge as a supporting medium in electrophoresis has been investigated. Segments of sponge were cut to fill horizontal and vertical cells which are described. The use of the described apparatus is recommended because of the ease of recovery of a product free from contamination and because of the latitude afforded in investigating migration through heterogeneous pathways. Thus, these cells were used in studies on zone electrophoresis and isoelectric migration employing serum, albumina nd preparations of human prostatic acid phosphatase. The applicability of the segmental sponge system to electrophoretic studies requiring either one or simultaneously several gradients such as density, pH and/or ionic strength has been demonstrated.

The further purification and properties of a phosphatase from spleen able to hydrolyze completely the phosphorus of α-casein

1. 1. The spleen phosphatase preparation of Singer and Fruton has been further purified using chromatography on triethylaminoethyl-cellulose columns and preparative electrophoresis. 2. 2. The purified enzyme moves as a single peak on electrophoresis in acetate buffer, pH 5.6, and has a blue-violet color. 3. 3. The enzyme is active toward phosphoramidate, p-nitrophenylphosphate, inorganic pyrophosphate and adenosinetriphosphate, but not towards glycerylphosphate or Ca(bis( p-nitrophenyl)phosphate) 2. 4. 4. The enzyme is able to remove completely the phosphorus of α-casein and of unfractionated “Hammarsten casein”.

Isolation of 32P-labeled Phosphoserine from a Preparation of Intestinal Alkaline Phosphatase Incubated with Radioactive Inorganic Phosphate.

Isolation of 32P-labeled Phosphoserine from a Preparation of Intestinal Alkaline Phosphatase Incubated with Radioactive Inorganic Phosphate.

The alkaline phosphomonoesterase activity of cell nuclei; activation by 0.16M magnesium.

I. The presence of 0.16M magnesium chloride in the incubation mixture for the Gomori method for localizing alkaline phosphatase activity, markedly increases the enzyme activity in the nuclei of the guinea pig endometrium, ovary and adrenal cortex, rendering them visible after 5 minutes' incubation. The activity in cytoplasmic and extracellular sites is decreased. The staining of these same nuclei could be demonstrated by the Loveless and Danielli method of visualizing the phenolic component of a complex phosphate ester instead of precipitating the phosphate ions with calcium salts. The activity described may be specific for the tissues mentioned and serves as an instance in which nuclear staining in phosphatase preparations is clearly valid. II. The effect of magnesium and cyanide ions and other factors on the enzyme activity noted histochemically in nuclei and biochemically in nuclear cell fractions, was reviewed. The data support the validity of some earlier histochemical observations describing a nuclear phosphomonoesterase activity in many organs which differs from that in cytoplasmic and extracellular sites. They also suggest that the phosphatase activity in cell fractions of those biochemical preparations prepared in aqueous media are altered by diffusion and adsorption phenomena.

The effect of zinc and magnesium ions on the activity of kidney alkaline phosphatase

1. 1. The effects of the addition of zinc and magnesium ions to cyanide-inhibited ox kidney phosphatase preparations have been studied. 2. 2. Zinc ions in low concentrations had a reactivating effect on the cyanide-inhibited enzyme, whereas magnesium ions had little effect. The effect of a mixture of these ions was dependent on their absolute amounts. 3. 3. Although there were evidences of slight tendencies for a shift of optimum pH on the addition of the metallic ions to the cyanide-inhibited enzyme, the changes were too small to support the general hypothesis of Sadasivan that the pH optima can be shifted by varying the ratios of zinc and magnesium ions. 4. 4. Comparison of the effects observed with ox kidney preparations and a human kidney phosphatase preparation was made.

Zone Electrophoresis of Intestinal Alkaline Phosphatase.

SummaryA crude preparation of alkaline phosphatase from bovine intestinal mucosa, obtained by treatment with butanol to release enzyme from the microsomes, was subjected to sequential electrophoresis on starch blocks. The results indicate that the phosphatase may have been present initially in a complexed form and that the complex was disrupted during repeated electrophoretic separation. Such behavior, together with the rather small amounts of enzyme protein which appear to be present in the starting material, may account for some of the difficulties encountered in attempts to purify intestinal alkaline phosphatase. An approximately 260-fold purification was achieved, but the amounts of final product obtained were too small for further study.

Histochemical and chemical evidence for more than one alkaline phosphomonoesterase.

A question exists se to whether all organic monophosphates are hydrolyzed by a single non-specific alkaline phosphatase or whether a number of such enzymes exist. Several histochemists have reported that the location and quantity of precipitate differs in alkaline phosphatase preparations when various substrates are compared (references, Gomori, 1952, p. 176). For the most part, these observations have been made on paraffin sections. However, recent appreciation of the occurrence of artefacts after paraffin embedding (e.g., Herman and Deane, 1953) has led to general skepticism that several phosphomonoesterases can be differentiated [although it is agreed that there is a specific enzyme capable of hydrolyzing adenosine-5-phosphate (Gomori, 1949)]. Using fresh frozen sections, Maengwyn-Davies and Friedenwald (1950) adduced evidence of the existence of a separate enzyme capable of splitting naphthylphosphate, differing from the enzyme or enzymes which hydrolyze aliphatic compounds. This conclusion has remained in question, however, since the periods of incubation were long and no cytological descriptions or ifiustrations were submitted. The present paper represents an attempt to study this problem in more detail, utilizing fresh frozen sections of a number of rat organs and of cat’sstomach. Gomori’s (1941) method was used for the demonstration of alkaline phosphatase with a ,j9-glycerophosphate, the precipitated calcium phosphate being visualized as cobaltous suffide.Manheimer and Seligman’s (1948) procedure was used with aand 9-naphthylphosphates, the released naphthol being converted to an insoluble azo dye with tetrazotized diortho-anisidine chloride (Diazo Blue B). For the naphthylphosphates, the site of hydrolysis was also confirmed by visualizing the phosphate as cobaltous sulfide. In addition, less extensive tests were carried out using fructose diphosphate, phenylphosphate, indoxyiphosphate and 6-benzoyl -naphthy1phospb.ate as substrates. The effects of various inhibitors and activators on the histochemical reactions of alkaline phosphatases with glycerophosphate and naphthylphosphates were investigated, and some of the results were checked by direct chemical means.

Coenzyme of kidney phosphatase.

IN previous communications1 we advanced a hypothesis supporting the nucleotide nature of kidney phosphatase. Kutscher and Schreier2 also consider phosphatases as metal proteins, and they attribute the loss in activity which they display on dialysis not only to the elimination of magnesium, but also to the separation of another substance which Kutscher and Sieg3 designate as choline pyro-phosphate. In their turn, Akamatsu and Kobayashi4 regard histidine as a coenzyme. Recently, however, investigating intestinal phosphatase preparations purified by paper electrophoresis, Roche considers5, in view of the results obtained in his analyses, that the hypotheses advanced on the nature of cophos-phatase (nucleotide, choline pyrophosphata or histidine) are scarcely probable.

The effect of molybdate on the activity of tomato acid phosphatases.

The effectiveness of sodium molybdate as an inhibitor of tomato acid phosphatases was examined. Significant inhibition of cell-free dialysed extracts was obtained with as low as 1O-7M sodium molybdate. A range of substrates, including a number of glycolytic intermediates, was tested and all except glucose I-phosphate were hydrolysed by tomato leaf phosphatase preparations. Hydrolysis was always inhibited by molybdate. The inhibition was competitive; Ki= IO-5M.

The path of carbon in photosynthesis. XVIII. The identification of nucleotide coenzymes.

The radioactive compounds to be observed when algae or green leaves are allowed to photosynthesize in C{sup 14}O{sub 2} for short periods are almost all phosphorylated derivatives of sugars. Of these, phosphate esters of trioses, sedoheptulose and fructose are the first to incorporate C{sup 14} followed closely by ribulose diphosphate, glucose-6-phosphate and a phosphate of mannose. It has been noted, in earlier papers of this series, that on radiograms of the products of photosynthesis, a dark area appeared in a position occupied by no known sugar phosphate and which gave glucose on acid hydrolysis or on treatment with a phosphatase preparation. This has hitherto been referred to as an 'unknown glucose phosphate'. It was found that this substance was more labile to acid than glucose-l-phosphate, itself a readily hydrolysable phosphate, and furthermore that other labile glucose derivatives were formed as intermediates during the acid hydrolysis. Accumulation of labeled glucose in this area precedes that in sucrose and suggests its synthetic relationship to sucrose phosphate synthesis.

Partial purification of bovine intestinal alkaline phosphatase.

SummaryA procedure has been presented for obtaining a soluble preparation of intestinal alkaline phosphatase from the microsomes of steer intestine mucosa by extraction of the microsomes with n-butanol. Although the purification achieved is only 15 to 25 times that of the original homogenate of the tissue, the resulting preparation appears to be particularly suitable for further purification by fractional precipitation with acetone.

Enzymatic dephosphorylation of triphosphopyridine nucleotide

Summaryo1.An enzyme preparation which hydrolyzes TPN to DPN and inorganic phosphate has been purified from pig kidney by ammonium sulfate fractionation and trypsin digestion.2.The enzyme activity is optimal at pH 9.5 and it is activated by Mg++ and to a lesser extent by Mn++ and Ca++. Rate studies showed that the reaction was of the first order.3.A method for the determination of TPN and DPN in mixtures in a single experiment is shown.4.The enzyme preparation was not specific for TPN and hydrolyzed several phosphate esters including adenylic acids a and b. Purified preparations of alkaline phosphatase showed high TPN-dephosphorylating activity. The relationship of the TPN-dephosphorylating enzyme to alkaline phosphatase is discussed. An enzyme preparation which hydrolyzes TPN to DPN and inorganic phosphate has been purified from pig kidney by ammonium sulfate fractionation and trypsin digestion. The enzyme activity is optimal at pH 9.5 and it is activated by Mg++ and to a lesser extent by Mn++ and Ca++. Rate studies showed that the reaction was of the first order. A method for the determination of TPN and DPN in mixtures in a single experiment is shown. The enzyme preparation was not specific for TPN and hydrolyzed several phosphate esters including adenylic acids a and b. Purified preparations of alkaline phosphatase showed high TPN-dephosphorylating activity. The relationship of the TPN-dephosphorylating enzyme to alkaline phosphatase is discussed.

Adsorption of bovine alkaline phosphatase on diatomaceous silica.

SummaryThe adsorption of a preparation of bovine alkaline phosphatase on Filter-Cel was investigated in respect to amount of adsorbent, concentration and kind of salt, pH and temperature. Calcined Celites were poor adsorbents for the phosphatase.

Obtaining uniform results in the histochemical technic for acid phosphatase.

The capricious nature of the results obtained in acid phosphatase preparations is notorious. An investigation of the causes of this is presented. It was found that it is possible to produce uniform preparations. In addition to many small details which must be carefully followed, two major factors radically affecting the results were observed: the technic of mounting the section on the glass slide, and the technic of deparaffinization and hydration. The final technic worked out is presented in the Summary and Conclusions.