Perry B. Hudson

Acid phoshatase. VI. Kinetic properties of purified yeast and erythrocyte phosphomonoesterase

Abstract Purified preparations of erythrocyte and yeast acid phosphomonoesterases were compared with respect to various kinetic properties including action on a variety of substrates, effect of activators and inhibitors, temperature effects, etc. Certain studies with purified prostatic acid phosphomonoesterase were also included for purposes of comparison. The erythrocyte and yeast enzymes were found to be extremely dissimilar in their properties. No greater similarity was in fact apparent between these as compared with the prostatic enzyme. It was concluded from these studies that in spite of previous classification of these two enzymes as similar types, (i.e., phosphomonoesterase, type IV) little if any basis could be found in support of such a scheme.

Acid phosphatase. III. Specific kinetic properties of highly purified human prostatic phosphomonoesterase.

Abstract The stability characteristics of highly purified prostatic acid phosphatase were investigated. The enzyme was found to be markedly susceptible to surface inactivation in dilute solution. The addition of suitable surface-active agents including synthetic nonionic detergents afforded complete protection during vigorous shaking of enzyme solutions. Surface inactivation was found to follow first-order kinetics. Enzyme-substrate constants ( K 8 ) were determined using four separate substrates. Methods for the reliable determination of these constants including the effect of ions were presented and discussed. The kinetics of enzyme inhibition by l -tartrate and oxalate were presented. Additional experiments relating to enzyme inactivation and reactivation studies by thiol reagents were also included as a part of this report.

Reduction of Postprostatectomy Bleeding by Epsilon-Aminocaproic Acid

EPSILON-aminocaproic acid (EACA) is a potent inhibitor of plasminogen activation in relatively low concentrations (5 × 10–4M) and of plasmin at considerably higher concentrations (1 × 10–2M).1 , 2 This drug, first described by Japanese investigators, has the structural formula: Clinical studies have indicated that it is effective in hemorrhagic disorders associated with increased blood fibrinolytic activity,3 4 5 and its use in prostatic surgery was first suggested by McNicol and his associates.6 These investigators suggested that hemostasis in the prostatic bed might be disrupted by clot lysis induced by the presence in urine of urokinase, an activator of plasminogen. Administration of .xa0.xa0.

Studies on the purification of acid prostatic phosphatase

Abstract 1. 1. Characteristics are given for facilitating 5000-fold purification of prostatic acid phosphatase. 2. 2. A purification starting with 40 human glands is described. 3. 3. Foaming as a purification technique is used and described. A relatively great purification is obtained with foaming. 4. 4. Experiments designed to demonstrate surface inactivation are presented. A study of methods of minimizing denaturation due to glass surface was made, and methods compatible with assay and isolation are presented.

Acid phosphatase. I. Human red cell phosphomonoesterase; general properties.

Abstract 1. 1. Phosphomonoesterase action of human erythrocytes was investigated using as substrates phenyl phosphate, α- and β-glycerophosphates, and yeast adenylic acid. Only one pH optimum of enzyme action (around pH 5.5) was observed with each of the substrates tested. 2. 2. Alterations in phosphomonoesterase activity were observed within certain hemolyzate preparations after aging (24 hr.), resulting in measured increases as well as decreases in enzyme action. 3. 3. Red cell phosphomonoesterase was activated by added magnesium (optimal activation at 0.01 M) in the presence of all substrates investigated over the entire active pH range. Neither prolonged dialysis or cold acetone (−10 °C.) precipitation of the hemolyzates resulted in enhancement of enzyme activation by magnesium. 4. 4. Optimal conditions for the accurate assay of red cell phosphomonoesterase were presented. 5. 5. A study of phosphotransferase action of red cell preparations was presented. On the basis of a theoretical treatment of the kinetics of transferase and phosphomonoesterase actions, the results are not incompatible with the view that both reactions are mediated through a single enzyme. 6. 6. The metabolism of adenosine by red cells was investigated and discussed in relation to the use of yeast adenylic acid as a substrate for phosphomonoesterase studies. Use was made in these investigations of phosphomonoesterase-free hemolyzates prepared with Fullers earth.

Acid phosphatase. IV. Fluoride inhibition of prostatic acid phosphatase

Abstract Inhibition of the acid phosphatase of human prostate by fluoride was shown to reach a maximum value at intermediate concentrations of fluoride and to be partially reversed by higher concentrations. The precise shape and location of the inhibition curve were influenced by ionic strength, by specific ions, and by protein concentration. A number of multivalent organic anions was shown to protect the enzyme against fluoride inhibition. The degree of protection afforded was greatest for anions such as citrate and oxalate. No evidence that this effect involves chelations of multivalent cations was found. A quantitative theory of these phenomena has been developed and shown to satisfy the experimental data, while some alternative theoretical possibilities have been eliminated or shown to be less plausible. The relation of these results to the structure of the active center of the enzyme is discussed.

Acid phosphatase. II. Purification of human red cell phosphomonoesterase.

Abstract A detailed investigation of calcium phosphate gels was carried out. It was concluded on the basis of these studies that the single most significant variable affecting the adsorption characteristics of calcium phosphate gels was the pH at which the gel was prepared. Gels prepared in the presence of sufficient NH 4 OH to give the reaction mixture a final pH 6–7 were found to be the best suited for the purification of the red cell phosphomonoesterase. Methods were described for the purification of red cell phosphomonoesterase by more than 1500-fold based primarily on the use of these gel preparations.

Some general characteristics of enzyme foam fractionation

Abstract Variations of foam purification conditions for urease and catalase, separately, were studied. It was demonstrated that impure urease and catalase mixtures could be separated considerably from one another, and each could be separated from most other contaminating protein. The effect of several variables on the foaming method of separating enzymes from protein was investigated, and some general rules were presented for the fractionation.

Acid phosphatase. V. The nature of inactivation and stabilization of purified human red cell phosphomonoesterase.

Abstract The stability characteristics of purified human red cell phosphomonoesterase were investigated. Two separate phenomena were identified to be primarily responsible for the instability of the purified preparation. The enzyme was found to be markedly susceptible to inactivation by surface forces. Kinetic studies relating to the surface inactivation process were presented. The addition of very small quantities of synthetic non-ionic detergents resulted in a complete stabilization of the enzyme against inactivation due to surface forces. In addition to the surface phenomenon, the enzyme was found to be rapidly inactivated by trace quantities of heavy metals which were present as a contaminant. Dialyzing membrane (cellophane as well as collodion) was found to contain sufficient quantities of heavy metal (iron, copper) to produce considerable inactivation of enzyme and was identified as the original source of these contaminants. The marked susceptibility of the enzyme to trace quantities of metals suggested its sulfhydryl nature. Evidence for the necessity of free —SH groups for the activity of this enzyme was presented.

Purification and properties of solubilized uricase.

Abstract A new method for preparing uricase is presented. The procedure is simple and yields the purest known enzyme. Advantage is taken of the affinity of the enzyme for other proteins in purifying it. The evidence indicates that the enzyme is an almost insoluble protein, and an explanation, based on its solubility properties, of the distribution of uricase in the cell is offered. Data on the variation of activity, stability and solubility as a function of pH are presented. The unusual appearance of the enzyme preparation is described.