Enzyme prostatic acid phosphatase

Babson proposed a-naphthyl phosphate as an essentially specific substrate for the activity of prostatic acid phosphatase in serum (104). However Marshall, Price, and Amador found that this substrate is not specific for the prostatic enzyme because urine of human females contain 50 times more acid a-naphthyl phosphatase than male serum and 50% as much activity as male urine. Platelets have significant activity and the serum activity can increase to abnormal values following clotting. These workers also observed elevated activities in females with skeletal metastases of the breast. In 50 hospitalized male patients who had no evidence of prostatic cancer and 25 hospitalized female patients, the incidence of false positive results was 12%, a magnitude sufficient to preclude meaningful clinical interpretation (105). 

Very recently, a sandwich assay for prostatic acid phosphatase antigen was carried out using two cascaded enzyme reactions to provide amplification of the immunochemical event. In one format, an optical readout was used whereby a forma-zan dye was generated by reaction of a dye precursor and NADH generated from the second enzyme cycle. In the electrochemical format, the NADH generated in the second enzyme cycle was used to reduce Fe(CN) to FeCCN) " which was then detected amperometrically. While the use of Fe(CN) in ECIA has appeared in the... 

AGIRE computer program for, 249, 79-81, 225-226 comparison to analysis based on rates, 249, 61-63 complex reactions, 249, 75-78 experimental design, 249, 84-85 inhibitor effects, 249, 71-75 potato acid phosphatase product inhibition, 249, 73-74 preliminary fitting, 249, 82-84 prephenate dehydratase product inhibition, 249, 72-73 product inhibition effects, 249, 72-73 prostate acid phosphatase phenyl phosphate hydrolysis, 249, 70 reactions with two substrates, 249, 75-77 reversible reactions, 249, 77-78 with simple Michaelian enzyme, 249, 63-71 [fitting equations, 249, 63] with slow-binding inhibitors, 249, 88 with unstable enzymes, for kinetic characterization, 249, 85-89. 

Although detailed structural as well as mechanistic knowledge of an enzyme is desirable, it is by no means necessary in order to design a suicide substrate. This has been shown by Myers and Widlanski (1993) who have designed a simple inhibitor of human prostatic acid phosphatase (PAP), an enzyme that is believed to be involved in the regulation of androgen receptor activity in prostate cells. Since the enzyme shows a preference for hydrolysis of aryl phosphates, the 4-(fluoromethyl)-phenyl phosphate (FMPP) was prepared as a substrate that would, on hydrolysis by the... 

Most investigators utilize p-nitrophenyl or a-naphthyl phosphate as substrate. The determination of serum prostatic acid phosphatase was developed by Fishman and Lemer (34) based on the d-(+)-tartrate inhibition of prostatic enzyme discussed below. Babson et al. (35, 36) demonstrated that a-naphthyl phosphate was much more easily split by prostatic than red cell phosphatase. Table V (35) shows the results obtained when prostatic or red cell phosphatase was added to human serum which had been incubated at pH 8.6 for 1 hr at 37° to destroy all endogeneous phosphatase activity. The table shows the superiority of a-naphthyl phosphate as substrate. 

Fig. 3. Surface inactivation rate of prostatic acid phosphatase by shaking and protection by added surface-active agent. Shaking mixtures (20 ml) contained purified enzyme (056 /ug of protein/ml) in 0.05 M acetate buffer at pH 5.5. The solutions were shaken in 50 ml volumetric flasks using a mechanical shaker (Burrell, model CC). Temperatures were maintained by immersion of the flasks in an appropriately set water bath. After specified intervals of shaking, duplicate 0.1 ml ahquots were removed into tubes containing Triton X-100. All tubes were assayed simultaneously, following the shaking procedure, with 0.05 M phenyl phosphate as substrate. Curve 1 Enzyme + Triton X-100 at 0°C and 29°C. Curve 2 Enzyme alone at 0°C. Curve 3 Enzyme alone at 29°C. From Tsuboi and Hudson (88).

Fia. 7. Inhibition of prostatic acid phosphatase by D-(+)-tartaric acid. The reaction mixtures all contained equivalent amounts of the enzyme preparation, the indicated concentration of substrate (pH 5.0), 0.05 iff acetate buffer (pH 5.0), and tartaric acid (pH 5.0) total volume, 4.5 ml. Each point represents average values of determinations made with 5 X 10-5 iff and 10 X 10"5 iff tartaric acid except in the case of /3-glycerophosphate for which 1 X 10 iff and 2 X 10 iff tartaric acid was used. From Kilsheimer and Axelrod (4 ). 

Prostatic acid phosphatase is partially and reversibly inactivated by calcium ion (45). Anions such as chloride, bromide, and thiocyanate inhibit prostatic acid phosphatase competitively with regard to substrate as well as noncompetitively. A kinetic analysis by London et al. (46) indicates that the noncompetitive inhibition was related to changes in charge on the protein molecule. A variety of nonspecific anions accelerate thermal denaturation of the enzyme. The enzyme is quite sensitive to a number of electrolyte changes, but it is not clear whether these factors are involved in biological control. 

Prostatic acid phosphatase is reversibly inactivated by p-mercuri-benzoate and by Cu2+ and Fe3+ (59). In contrast to red cell acid phosphatase, prostatic acid phosphatase is only partially inactivated even after prolonged periods of incubation at high concentrations of p-mercuri-benzoate. Addition of cysteine to the p-mercuribenzoate-treated enzyme produces complete reactivation. Binding of SH groups by p-mercuri-benzoate renders the enzyme more labile to thermal denaturation, but no difference is obtained with surface inactivation (23). Similar partial inactivation with Cu2+ is also subject to reactivation. 

The incubation digest (7.0 ml) contained 1 ml of 0.022 M phenyl phosphate 2.5 ml of 0.1 M acetate buffer, pH 5.0 0.5 ml of test enzyme solution and 3.0 ml of solutions of acceptors giving a final concentration as shown in the third column. Incubation time, 30 min. Digests were inactivated by 3.0 ml of 10% trichloroacetic acid solution and were analyzed for phenol and inorganic phosphate. In the case of the standard acceptor, 1,4-butanediol, the expected transfer product, 1,4-butanediol phosphate, was isolated in a yield of 35% from a large-scale experiment. The hydrolysis of this phosphate ester by prostatic acid phosphatase liberated approximately equimolar amounts of 1,4-butanediol and inorganic phosphate. 

Shaw (115) reported a 300-fold purification of enzyme from tobacco leaves. Activity of the enzyme was optimal at pH 5.5-5.7, and divalent cations were not required for activity. The enzyme possessed high activity toward ribonucleoside 2 - and 5 -monophosphates and glucose 1-phosphate. There was no activity toward RNA or phosphodiesters. Fluoride acts as a noncompetitive inhibitor for this enzyme. This behavior of fluoride is in contrast to the behavior with prostatic acid phosphatase where the inhibition is strictly competitive. 

Putrefactive hacteria, such as Clostridium welchii, which frequently invade human blood during the agonal period or immediately after death, produce the enzyme neuraminidase ( 9). Neuraminidase has been shown to effect the heterogeneity of electrophoretic banding patterns of the human prostate acid phosphatase (10). The effect of this enzyme on EAP is not known. 

The preceding description of the use of chromatographic methods in the purification of prostatic acid phosphatase (B24, 04) has already indicated that this enzyme exists in more than one molecular form, or isoenzyme. There is, in addition, immunological (S19) and starch gel electrophoretic evidence (L14, L15, S24, S31) of the existence of several forms. In order to ensure that no isoenzymes are lost during any purification, it is preferable to perform such studies on a homogenate of the whole tissue. It should be recognized that the isoenzymatic composition may not be characteristic of the prostatic cell per se, but may also represent components from blood cells, secretory ducts, connective tissue, and other sources. 

Another procedure to increase the specificity of acid phosphatase determinations for prostatic disease has involved the use of n- (-I-) -tartrate to distinguish between the enzyme from the prostate and other tissues. In a series of papers from 1947 to 1949, Abul-Fadl and King (Al, A2, A3, A4) studied the properties of various acid phosphatases and reported that 0.01 Af L- (4-) -tartrate inhibited the hydrolysis of phenyl phosphate by human prostatic acid phosphatase dissolved in normal saline or in plasma to the extent of 95%, but had no effect on the hydrolysis by acid phosphatase from erythrocytes. The inhibitions of acid phosphatases from other human tissues were as follows liver, 70% kidney, 80% spleen, 70%. 

Alkaline phosphatase is one of the most suitable enzymes for electrochemical immunoassays owing to its high turnover number and broad substrate specificity. Different substrates have been used, but 4-aminophenyl phosphate is most suitable, since the reaction product, 4-aminophenol is easily oxidized without fouling of the electrode surface. Thyroxine-binding globulin, cortisol, and prostatic acid phosphatase have been detected by using alkaline phosphatase. 

Acid phosphatases are enzymes that have been studied extensively due to the fact that their dysregulation is associated with pathophysiological conditions. This characteristic has been exploited for the development of diagnostic and therapeutic methods. As an example, prostatic acid phosphatase was the first marker for metastatic prostate cancer diagnosis and the dysregulation of tartrate resistant acid phosphatase is associated with abnormal bone resorption linked to osteoporosis. 

The pioneering crystallization smdies on prostatic acid phosphatase and mammalian tartrate-resistant acid phosphatase conformed significant milestones towards the elucidation of the mechanisms followed by these enzymes (Schneider et al., EMBO J 12 2609-2615, 1993). Acid phosphatases are also found in nonmammalian species such as bacteria, fungi, parasites, and plants, and most of them share structural similarities with mammalian acid phosphatase enzymes. 

The lysosomal acid phosphatase enzyme played a key role in the discovery of lysosomes by de Duve in 1963 and is widely used as a lysosomal marker. This enzyme shows a high degree of sequence similarity (ca. 49 % identity) with prostatic acid phosphatase [9] and both are inhibited by L-(+)-tartrate ion [10]. 

In 2007 we characterized a novel transmembrane type-I isoform of the prostatic acid phosphatase enzyme (TMPAP) as the product of a splice variant of the same gene encoding the secreted form (sPAP). This transmembrane type-I isoform contains a tyrosine-based lysosomal targeting (YxxO) motif at the... 

All these significant physiological findings must be taken into account during the evaluation of inhibitors and activators of prostatic acid phosphatase enzyme for therapeutic use. 

Prostatic acid phosphatase (EC 3.1.3.2) can be purified from seminal fluid, prostate tissue, or as a recombinant protein. High-scale puriflcation methods are important in order to obtain mass amounts of homogeneous, purifled protein required by structural and functional studies such as inhibitor and activator analyses. Acid phosphatases can be divided into two groups according to their sensitivity or resistance to L-(-i-)-tartrate inhibition. The activity of these enzymes can be analyzed by the -nitrophenyl phosphate method in presence [1] or absence of L-(-i-)-tartrate [2]. PAP belongs to the group of L-(-i-)-tartrate sensitive acid phosphatases, and this feature is used as the basis of the puriflcation method described in this chapter. 

V6. Vihko, P., Human prostatic acid phosphatases. Purification of a minor enzyme and comparisons of the enzymes. Invest. Urol. 16, 349-352 (1979). 

Immunological methods for enzymes, more specifically isoenzymes, such as lactate dehydrogenase-1 (167, 168), mitochondrial aspartate aminotransferase (169), prostatic acid phosphatase (170, 171,172), and creatine kinase-MB (173, 174, 175), have been in use in the clinical laboratory for 10 years. However, the use of the immunological rather than catalytic properties of enzymes has not provided the opportunities for standardization that was anticipated a number of years ago (176, 177, 178). It is only within the last year that a working group on CK-MB mass assay was formed under the auspices of the Standards Committee of the American Association for Clinical Chemistry (AACC). The objective of this working group is to prepare a reference material to calibrate methods that are based on the principle of CK-MB mass measurement. 

Tumour markers fall into one of several groups they may be homiones. e.g. human chorionic gonadotrophin (HCG) secreted by choriocarcinoma or enzymes, e.g. prostatic acid phosphatase in prostate carcinoma or tumour antigens. e.g. carcinoembryonic antigen (CEA) in colorectal carcinoma. 

A minor isoenzyme of human prostatic acid phosphatase (pi 5.5) has been separated from the major isoenzyme (pi 4.9). Immunological similarities between the two enzymes were demonstrated. 

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