William H. Fishman

Human serum β-glucuronidase; its measurement and some of its properties

Abstract Human serum β-glucuronidase has a high requirement of phenolphthalein β- d -glucosiduronic acid for its saturation as evidenced by the apparent K m value of 0.002 M . The digest containing 0.006 M substrate ensures an elevated linear rate as a function of serum concentration which permits the reduction of incubation time to 4 h. One can expect a variation of between 1.4 and 6.5% in replicate determinations depending on the enzyme level. The role of circulating saccharolactone, an endogenous β-glucuronidase inhibitor, was found to be minimal following the study of the effect of dialysis on enzyme activity. Serum values in normal individuals and in patients with diabetes, liver cirrhosis and cancer are reassessed and were found to be approximately twice the previously reported values. Conditions have also been recommended for the use of alternate substrates, i.e. β- d -glucosiduronic acids of p -nitrophenol and 8-hydroxyquinoline. Finally, factors of biochemical specificity, of integrity of subcellular organelles and genetics are dealt with in the evaluation of the significance of serum β-glucuronidase values.

Isoenzymes of Human Alkaline Phosphatase

Publisher Summary Isoenzyme refers to a biochemically distinct class of catalytically active proteins with the same specificity of bond cleavage or alteration, which can occupy several zones following electrophoresis. The chapter focuses on the mechanism of catalysis and uncompetitive inhibition and provides analyses of studies reporting distinctive immunochemical properties as well as physical properties of enzymes prepared from several organs. In the case of alkaline phosphatase, the studies rely on a combination of organ source, biochemical properties, and electrophoretic distinctions as guidelines for working in this field. Placental alkaline phosphatase is an isoenzyme of alkaline phosphatase, which can be distinguished from other alkaline phosphatases by biochemical means. The different molecular forms of this isoenzyme, which can be produced and separated by physical means, are referred as variants of placental alkaline phosphatase. Hyperphosphatasemia in diseases of liver and bone has made the serum alkaline phosphatase determination the most frequently demanded enzyme assay.

New observations on the Regan isoenzyme of alkaline phosphatase in cancer patients

The Regan isoenzyme (RI) of alkaline phosphatase is interesting because it is indistinguishable from the placental isoenzyme with regard to stability to heat, inhibition by L‐phenylalanine, optimum pH. and specific reaction with rabbit antisera to placental alkaline phosphatase. The current study of 323 cancer patients was conducted at a general hospital where cancer is more frequently seen at an earlier stage of natural history and therapy than in a chronic disease hospital. A positive identification of RI was made in 39 patients (12%) by biochemical and immunologic techniques, including antibody neutralization and starch gel electrophoresis. A high incidence of carcinoma of the ovary (5 of 23) and seminoma (one of one) patients contrasted to a somewhat lower incidence in bronchogenic carcinoma (7 of 51) and breast cancer (6 of 49). Most interesting was the discovery of high RI levels in familial polyposis of the colon, and ulcerative colitis, diseases with a known cancer diathesis. In general, RI dropped sharply following treatment with effective antimetabolic chemotherapeutic agents. The ectopic production of this protein enzyme, interpreted as a consequence of derepression of the tumor cell genome, is comparable with other polypeptide hormone syndromes, or carcinoembryonic antigens, but evidences a wider variety of sites of tumor origin. Its identification in gonadal tumors and in precancerous states may represent a predeliction of such derepression for tissues concerned with germinal activity or with genetically determined cancer diathesis, respectively.

Organ-specific behavior exhibited by rat intestine and liver alkaline phosphatase.

Abstract Some 130 compounds have been evaluated for their effect on the behavior of rat alkaline phosphatase present in liver, intestine, bone, lung, kidney and blood serum in the presence and in the absence of Mg 2+ . Intestinal alkaline phosphatase could be inhibited in a specific manner by substances such as dl -phenylalanine, dl -leucine and glutathione and was not inhibited specifically by substances such as Zn 2+ , taurocholate, hyocholate which, however, inhibited the enzyme of the other tissues. Inhibitors which specifically spared liver alkaline phosphatase were CN − , thioglycolate, S -carbamyl cysteine and l -cysteine ethyl ester. Organ specificity was noted in the hydrolysis of orthophosphoric acid monoesters. Thus, phosphoethanolamine and menthol phosphate were preferentially hydrolyzed by liver alkaline phosphatase and o -carboxyphenylphosphate by intestinal alkaline phosphatase. By employing a combination of organ-specific reagents and “preferential” substrates, it was possible to obtain satisfactory recovery of intestine and liver alkaline phosphatase from mixtures of alkaline phosphatase prepared from 9 different organ sources. The behavior of normal rat serum alkaline phosphatase in all these experiments was indistinguishable from the intestinal enzyme which supports earlier views of the intestinal origin of the serum alkaline phosphatase.

Serum alkaline phosphatase of intestinal origin in patients with cancer and with cirrhosis of the liver

Abstract A study has been completed on the l -phenylalanine-sensitive alkaline phosphatase in sera of normal subjects, of cancer patients and of patients with cirrhosis of the liver. It has been previously established that only intestinal alkaline phosphatase is inhibited by l - and not d -phenylalanine. In normal subjects, some 40% of the serum alkaline phosphatase is composed of enzyme contributed to by the intestine. The upper value for this component was found to be 3.00 units. Patients with cancer evidence a lower proportion than normal of the contribution of intestinal alkaline phosphatase to the total serum value. The elevated total alkaline phosphatase values encountered in such patients must represent contribution to the serum level from tissues other than intestine. Subjects with cirrhosis of the liver show a normal average but a wide range of contributions (10–80%) of the intestinal component. In 10 out of 33 patients with cirrhosis of the liver, the intestinal alkaline phosphatase exceeded 3.4 units, and was 8.3 units in one individual. In 8 of these 10 patients, portal hypertension was obviously present. It is now possible to divide patients with liver disease exhibiting an abnormally elevated alkaline phosphatase into those which can be explained by a high l -phenylalanine-sensitive alkaline phosphatase and into those which cannot. It is further necessary to recognize the quantitative importance of the contribution of the intestinal source to the total alkaline phosphatase value in health and disease, in general.

Identification by means of L-phenylalanine inhibition of intestinal alkaline phosphatase components separated by starch gel electrophoresis of serum

After electrophoresis of serum on starch gel, the alkaline phosphatase-rich zones which undergo inhibition by l-phenylalanine, a specific inhibitor for the intestinal enzyme, have been clearly defined and quantitatively measured. Serum from normal subjects, with a major peak of alkaline phosphatase activity in the fast α2 region, shows some intestinal alkaline phosphatase on the trailing edge of the peak. Occasionally a second intestinal peak is observed in the haptoglobin region, where alkaline phosphatase activity of intestinal tissue extracts is found7. This is also the location of an intense activity present in the serum from some cirrhotic patients. Another cirrhotic with a normal percentage of intestinal component but with highly elevated alkaline phosphatase total, exhibited no haptoglobin peak. These subjects are examples of two categories of cirrhotic patients with elevated serum alkaline phosphatase6, one in which the intestinal component is predominant and one in which it is not.

Dual localization of acid hydrolases in endoplasmic reticulum and in lysosomes

SummaryDissimilar enzyme locations obtained on occasion by the post- and simultaneous-coupling techniques employing the substrate naphthol AS-BI β-glucosiduronic acid were attributed to the inadequate incorporation of substrate into lysosomal membranes in the post-coupling technique on the one hand, as well as to the inhibition of cytoplasmic enzyme by diazotate in the simultaneous coupling technique on the other hand. The use of a fixative solvent mixture prior to the enzyme staining reaction appeared to labilize lysosomal membranes, to improve fixation and to eliminate “fiber” artefacts. In male mice which have been androgenized by the injection of gonadotrophin, kidney homogenates, subsequently prepared, exhibited an immediate increase in the specific activity of microsomal β-glucuronidase while lysosomal β-glucuronidase was unchanged for the first 36 hours.This event at 36 hours corresponded with enhanced cytoplasmic but not lysosomal staining. “Diffuse reactions” in enzyme morphology are discussed as well as the origin of lysosomal β-glucuronidase in mouse kidney and the dual localization of hydrolases in endoplasmic reticulum and lysosomes.

β–GLUCURONIDASE AND THE ACTION OF STEROID HORMONES

It may be desirable a t the outset to define the goals of research in the field of enzymes and the steroid hormones. In the first place, it is understandable that the explanation of the biological action of the sex steroids should be sought in the realm of enzymatic phenomena. Thus, as we know, the generalization that most biochemical reactions in the living organism are catalyzed by specific enzymes is widely held and accepted. Moreover, the success which has been realized in establishing the participation of various vitamins in essential specific enzyme systems of the cell has, in turn, led to the expectation that the steroid hormones may similarly be shown to be components of important enzyme systems. General Considerations. First, some of the concepts will be outlined which may apply to the participation of steroids in tissue enzyme systems. Reference will be made only to the estrogenic hormones presented in FIGURES 1 and 2. FIGURE 1 is a simplified picture of estrogen metabolism which indicates the main pathways of estrogen in the body. As can be seen, two forms of estrogen occur in the body, free and conjugated. The free estrogen may either be excreted mostly in the bile or be degraded oxidatively in the liver by non-specific oxidases. The conjugate of estrogen (either its glucuronide or sulfate) is excreted in the urine. Both free estrogen and its conjugates will stimulate growth processes in the secondary sex tissues. Accordingly, it is felt that the explanation of the action of the estrogens in terms of enzyme phenomena should be sought in these mechanisms labeled A and B, which may possibly be one and the same process. The utilization of estrogen as an integral component of an enzyme system is most simply represented in FIGURE 2. Enzyme X, which is the term signifying that the enzyme is hypothetical and as yet unidentified, should be the limiting factor in tissue growth stimulated by estrogen. If we may reason by analogy from experiences in the enzyme-vitamin field, all vitamin coenzymes which have been studied thus far are acidic in nature. It may not be too unreasonable to predict that, should the sex steroids be found to constitute coenzymes, these hormones will possess acid functions. Estrogen may participate in an enzyme system as a specific substrate, as in FIGURE 2. Perhaps the enzyme-substrate complex has a physiological function in growth which is distinct from its enzymological significance. It is necessary in a field as new and as confused as that constituting steroid-enzyme phenomena to distinguish between primary and secondary enzyme reactions resulting from hormonal action. A primary enzyme reaction of hormone-induced growth, in this regard, may be defined as one in which the steroid participates directly, either as coenzyme, substrate, activator, or inhibitor. Accordingly, secondary enzyme reactions of hor-

Distinctions between intestinal and placental isoenzymes of alkaline phosphatase

Abstract 1. 1. Placental and intestinal alkaline phosphatases are equally inhibited by 0.005 M l -phenylalanine, a fact which complicates the interpretation of the values for l -phenylalanine-sensitive alkaline phosphatase of pregnancy serum which can be expected to have both phosphatases. 2. 2. Contrary to (human and rat) intestinal alkaline phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) the isoenzymes of human and rat placental tissues are split by neuraminidase ( N -acetyl neuraminate glycohydrolase, EC 3.2.1.18) i.e. the anodic mobilities of the placental enzymes are appreciably reduced by prior incubation with neuraminidase. 3. 3. Advantage is also taken of the marked heat-stability of placental alkaline phosphatase and thus starch gel electrophoretic patterns of the placental and intestinal enzymes in heated and unheated specimens are different from each other. 4. 4. The pH optima of human intestinal and placental alkaline phosphatases are 9.8 and 10.6 with 18 m M phenylphosphate as substrate. The ratio of the enzyme activity at pH 10.6 to that at pH 9.8 is ten times higher for placenta than that for intestine (1.1 versus 0.11) with 2 m M phenylphosphate as substrate. 5. 5. Although the Michaelis constants of placental and intestinal alkaline phosphatase at pH 10.6 are the same, the K m -values of placental alkaline phosphatase at lower pHs are considerably higher than those of the intestinal isoenzymes. 6. 6. The above electrophoretic and kinetic differences may be employed to distinguish from each other human intestinal and placental alkaline phosphatase isoenzymes and may be utilized further to investigate the intestinal and placental origins of serum alkaline phosphatase.

Dual localization of β-glucuronidase and acid phosphatase in lysosomes and in microsomes

SummaryThe dual localization of certain hydrolases in lysosomes and in endoplasmic reticulum as studied in enzyme staining reactions is now supported by cytobiochemical studies on mouse liver and kidney β-glucuronidase and acid phosphatase. Use was made of the renal β-glucuronidase response to endogenous androgen for both studies. Accordingly, sucrose homogenates were prepared of liver and kidney of male BALB/C mice previously injected with gonadotrophin along with control animals receiving saline instead. The homogenates were subjected to differential ultracentrifugation yielding six fractions. These were characterized as to their organelle composition by measurements of “marker” enzymes and by observations with the electron microscope. In all subcellular fractions, β-glucuronidase was uniformly increased 5 to 8 times over the corresponding control value and, in fractions rich in lysosomes, this enzyme was easily released by alternate freezing and thawing. On the other hand, the microsomal β-glucuronidase and acid phosphatase enzymes were not liberated by freezing and thawing nor were they after treatment with 0.1 % Triton X-100 and by employing other reagents and conditions which are known to release lysosomal enzymes. In contrast to microsomal acid phosphatase, microsomal β-glucuronidase activity could be liberated by treatment with hyaluronidase. This soluble β-glucuronidase showed the same optimum pH, Michaelis Constant and heat inactivation behavior as the lysosomal β-glucuronidase prepared by freezing and thawing treatment. These observations define two populations of microsomal vesicles each identifiable by an individual membrane-associated acid hydrolase. One of these β-glucuronidase, increases in specific activity in the animal on androgens and is released by hyaluronidase and the other, acid phosphatase, does not respond to androgen and is not released by hyaluronidase. There would appear to be a variety of mechanisms by which hydrolases enter into association with the membranes of the endoplasmic reticulum and from there, a variety of routes to the lysosomes. A comment is made concerning the question of acid phosphatases and β-glucuronidase as enzyme markers for lysosomes in mouse kidney.