Cytochemistry and biochemistry of acid phosphatases

Summary Acid phosphatases of the rat ventral prostate were studied cytochemically using different substrates. The results were compared to findings on isoelectric focussing gels stained for acid phosphatase activity. This is a highly specific and reproducible method which allows the distinction between secretory androgen-dependent and lysosomal acid phosphatases. Activity of lysosomal acid phosphatase was increased after castration, while the activity of the secretory enzyme gradually decreased after androgen deprivation. None of the substrates tested was selectively hydrolyzed by either secretory or lysosomal acid phosphatase. Phenylphosphate, creatine phosphate and choline phosphate were found to be inappropriate substrates for histochemical purposes, however, reproducible results were obtained with -naphthylphosphate, -glycerophosphate and p-nitrophenylphosphate. The method of isoelectric focussing (pH range 4.0–8.0) of enzymes with subsequent histochemical staining demonstrated lysosomal enzymes at pH 7.9 and 8.2 respectively. Small amounts of identical enzymes were found in liver, kidney, blood or epididymis. Secretory acid phosphatases were focussed at pH 5.5, 5.6, 5.65 and 7.15. Similar enzymes have been identified in epididymis, kidney, liver and pancreas. These results indicate that 1) at present no specific substrate for prostatic secretory or lysosomal acid phosphatases is available and 2) that no prostate-specific prostatic acid phosphatase (PAP) exists in the rat ventral prostate.Supported by the Deutsche Forschungsgemeinschaft (Au 48/6)     

Purification and partial characterisation of tentatively classified acid phosphatase from the earthworm Eisenia veneta

In order to use leakage of lysosomal acid phosphatase (AP) as a biomarker of stress to earthworms, more information about AP’s in earthworms are needed. This paper describes the details about tentatively classified APs in the earthworm Eisenia veneta. Two isoenzymes (enzyme I and II) of acid phosphatase (AP) and one alkaline phosphatase (enzyme III) from the earthworm E. veneta were separated by gel filtration. All three enzymes were further purified and concentrated on a Con A Sepharose 4B column. Enzyme I was inhibited by tartrate, showed an optimal pH range between 4.0 and 5.0 and was assumed to be of lysosomal origin. Enzyme II was the major enzyme showing the highest activity of the three enzymes. It was expected to be a lysosomal AP under physiological conditions. Enzyme II had a molecular mass 113 kDa and was composed of apparently identical polypeptide chains of 36 kDa each. This enzyme was inhibited by tartrate, showed an optimal pH in the range 6.0–7.5 and was slowly degraded at temperatures above 40°C. Enzyme III is not inhibited by tartrate and has a pH-optimum >9. The subcellular location under physiological conditions was assumed to be the cytosol.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

Human liver acid phosphatases.

Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively.Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline.     

Multiple forms of acid phosphatase in rat liver parenchymal,endothelial, and kupffer cells

Purified suspensions of highly viable parenchymal, endothelial, and Kupffer cells were prepared from rat liver. In the liver cell classes, total activities of acid phosphatase were determined with 4-methylumbelliferylphosphate, 1-naphthylphosphate, and p-nitrophenylphosphate. The specific enzyme activities were different for each type of cell and, even within one cell class, the enzymes showed different conversion rates for the three substrates. These results indicate the presence of multiple forms of acid phosphatase enzymes in each cell class. The inhibiting effects of tartrate, fluoride, and alloxan on the acid phosphatase activities were investigated. Depending on the substrate used, the inhibitors inactivated the enzymes at different rates, which also indicates the presence of multiple forms of acid phosphatase enzymes in the liver cell classes. By means of an isoelectric focusing technique, acid phosphatase enzymes could be separated on the basis of their differences in isoelectric points. One form with an isoelectric point around 4 is found in Kupffer cells, whereas another form with an isoelectric point of about 7 is found in parenchymal cells. Endothelial cells possess both forms. These findings suggest a specificity in the function of this lysosomal enzyme in each cell class.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl posphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at near-neutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membranes. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.     

Regulation of bile acid synthesis. Isolation and characterization of microsomal phosphatases

In preliminary experiments, we have shown that rat liver microsomes possess phosphatase activity which was inhibited in the presence of sodium fluoride. We have now separated six microsomal phosphatase fractions appearing to be isoenzymes. They all possess different kinetic constants and are not equally inhibited by tartrate and fluoride ions, inhibitors of phosphatase activity. One phosphatase fraction, in fact, is almost completely unaffected by fluoride ion. More pertinent to our interest, these isoenzymes exhibit differing abilities to modulate the activities of hydroxymethylglutaryl CoA reductase, acyl-CoA:cholesterol O-acetyltransferase, and cholesterol 7 alpha-hydroxylase. Interaction of four of the fractions with rat liver microsomes resulted in a decrease in cholesterol 7 alpha-hydroxylase activity; two were without effect.     

Zinc activated tartrate resistent phosphatases in the brains of different animal species and their characterization]

A zinc activated tartrate resistent phosphatase (ZnTP) of the brain of different animal species was separated by electrofocusing in polyacrylamide gels. It is demonstrable selectively in the presence of 20 mM zinc acetate and 10 mM D,L-sodium tartrate or of 100 mM zinc acetate only. The ZnTP hydrolyzes 1-naphthyl phosphate and 4-nitrophenyl phosphate, respectively. High activity of ZnTP is evident in the brains of rats and rabbits. The activity is moderate or absent in the brains of mice, syrian-hamsters, sheeps, cats, rhesus monkeys, and of human beings. The isoelectric points of the enzyme from the various species are different, but the molecular weight is identical (65 000 estimated by gelfiltration on Sephadex G 100 in the brain of rat, rabbit, syrian-hamster, and man). A method of quantitative evaluation of ZnTP activity is described.     

Stable acid phosphatase: II. Effects of pH and inhibitors

Summary Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl phosphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at nearneutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membrances. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.Supported by the Medical Research Council of Great Britain     

Ultrastructural demonstration of phosphatases by perfusion fixation followed by perfusion incubation of rat liver.

An alternative to previous methods (tissue chopper, frozen sections) for the ultrastructural demonstration of phosphatases is described. The present approach is based on a short vascular perfusion of rat liver with glutaraldehyde through the inferior caval vein, followed by vascular perfusion incubation with a medium containing the enzyme substrates. The effect of glutaraldehyde on three different types of phosphatases was investigated, namely a lysosomal enzyme (acid phosphatase) a tightly bound microsomal enzyme (G6Pase) and a loosely bound microsomal enzyme (IDPase). It is demonstrated that by perfusion with glutaraldehyde for three minutes good cellular morphology is obtained and that 50-60% of the initial activity of glucose-6-phosphatase, inosine-diphosphatase and acid phosphatase remains. The localization and deposition of G6Pase activity were distinct and observed throughout the endoplasmic reticulum and the nuclear envelope. For acid phosphatase, the reaction product was confined to various types of lysosomes including presumed autophagic vacuoles. No signs of enzyme diffusion were noted. The present approach seems to offer some advantages: it is simple and requires no extra equipment, penetration of the fixative and incubation enzyme medium is good, and finally freeze artifacts are avoided.     

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes.

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P20,2) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid beta-glycerophosphatase (EC 3.1.3.2) and acid p-nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p-nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1, 1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1, 1-diphosphonate had any effect on beta-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1, 1-diphosphonic acid inhibited acid p-nitrophenyl phosphatase strongly, the polyphosphate (P20, I) had little effect. Acid p-nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1, 1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.     

An isoelectric focusing study of acid phosphatase heterogeneity in rat liver

The different forms of acid phosphatase (EC 3.1.3.2) in rat liver homogenates, lysosomal, mitochondrial, microsomal fractions and cytosol were studied with isoelectric focusing. Evidence is presented that isoelectric focusing of acid phosphatase in subcellular fractions shows individual changes and time related patterns. Mild autolysis shifted all enzyme activity peaks of isoelectric focusing patterns to the one at pH 7.04.     

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r     

Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum

Two types of extracellular acid phosphatases are synthesized by Aspergillus ficuum NRRL 3135: a nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with an optimum pH of 2.0, and an enzyme with restricted specificity, a mesoinositol-hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with an optimum pH of 5.5. Although the pH 5.5 enzyme is termed a phytase, both enzymes hydrolyze phytin. Synthesis of the enzymes is repressed by high orthophosphate concentrations in the fermentation medium. The highest total level for each enzyme is synthesized in low orthophosphate medium. In high orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme. In low orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme during the early stages of growth, but the reverse occurs after 5 days. The enzymes are differentiated by heat denaturation at acid and alkaline pH levels. They are separated into two distinct fractions on Sephadex G-100 followed by carboxymethylcellulose column chromatography. This indicates that the two enzymes are structurally different. The K(m) for both enzymes is 1.25 mm when calcium phytate is the substrate. Orthophosphate competitively inhibits the pH 2.0 (K(i) = 1.1 x 10(-2)m) but not the pH 5.5 phosphatase. Neither enzyme is denatured by 50% (w/v) urea or inhibited by 0.01 m tartrate. Thus, they differ from human prostatic phosphatase.     

Histochemical identification of osteoclasts. Review of current methods and reappraisal of a simple procedure for routine diagnosis on undecalcified human iliac bone biopsies

Osteoclasts are known to have a high acid phosphatase content. We have adapted the simple simultaneous mono-coupling azo-dye method of Grogg and Pearse to undecalcified bone sections. A cold embedding in a mixture of glycol and methyl methacrylate was shown to well preserve the enzyme activity. Sodium alpha-naphtyl phosphate (1 mg/ml) and fast violet B (2 mg/ml) are used in 0.1 M acetate buffer, pH 5.0. The addition of 1 mM L(+) sodium tartrate selectively inhibits the acid phosphoprotein phosphatase ("osteoblastic acid phosphatase") but not osteoclastic lysosomal acid phosphatase. Counterstaining with phosphomolybdic aniline blue WS leads to well contrasted sections, providing accurate measurements of osteoclast number.     

Detection of alkaline phosphatase activity after conventional isoelectric focusing by an indoxyl-tetrazolium salt technique

Alkaline phosphatase was solubilized from human and rat tissues using papain in the presence of TRITON X-100 and subjected to isoelectric focusing (IEF) in polyacrylamide or agarose gels. Up till now, usually 1- and 2-naphthylphosphates have been used as substrates in order to specifically stain molecular forms of this enzyme by the azo-dye technique. In this paper, the use of another histochemical substrate, 5-bromo-4-chloro-3-indoxyl phosphate, in combination with tetrazolium salts [McGadey, J. (1970) Histochemie 23, 180-184] is presented. After hydrolysis, the released indoxyl moieties reduce tetrazolium salts to insoluble formazans at the zones of alkaline phosphatase activity. Zymogrammes showing molecular forms of alkaline phosphatase from 20 rat organs and the application of this staining technique for the detection of alkaline phosphatase activity in non-dialyzed human plasma after IEF are presented.     

Alkaline phosphatases of the mosquito, Culex tarsalis Coquillett

Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the alkaline phosphatase (ALKP) of the mosquito, Culex tarsalis, are presented. With p-nitrophenylphosphate (Pnp) as substrate, ALKP was optimally active at 37 degrees C, pH 8.0, 30 mM MgCl2, Vmax was 35.8 mumoles/10 min and the Km was 5.7 mM, with no demonstrable requirement for Zn2+. The spectrophotometric enzyme(s) was stimulated by dithiothreitol, 2-mercaptoethanol, and poly-vinylpyrollidone (PVP); inhibited by NaF, several alternative cations (Ca2+, Ba2+, Fe2+, Cu2+), and EDTA. ALKP activity was cyclic during the 15 day post-adult emergence period of the study. No significant differences were noted between the specific activities of males and females. IEF electrophoresis revealed 6 ALKP isozymes detected with alpha-naphthylphosphate within the pH range 4.0-5.5, with a second group of 3 rather indistinct species in the pH 6.0-7.0 range. IEF ALKP isozymes were stimulated by Mg2+ and PVP and inhibited by EDTA (except ALKP5.0) and cysteine; partial inhibition with phenylalanine. IEF detection of ALKP activity with Pnp indicated that the majority of the activity was localized in the pH 4.0-5.5 range, in close agreement with the alpha-naphthylphosphate results.     

Direct tissue isoelectric focusing on mini ultrathin polyacrylamide gels followed by subsequent western blotting, enzyme detection, and lectin labeling as a tool for enzyme characterization in histochemistry.

Application of cryostat sections directly onto mini ultrathin polyacrylamide isoelectric focusing gels allows an elution of proteins out of the sections into the gels under conventional focusing conditions. Protein bands representing alkaline phosphatases can easily be identified on nitrocellulose after performing a modified Western blot procedure. Furthermore, carbohydrate residues of several isoforms of alkaline phosphatases separated by isoelectric focusing can be demonstrated in a single blot strip by subsequent incubation of this strip with substrates for alkaline phosphatase and peroxidase, the latter being employed as the enzyme to which the applied lectins are covalently linked. This simple and reproducible procedure is likely to enable histochemists to determine isoforms of enzymes from a single cryostat section.     

Conversion of skeletal tartrate-sensitive acid phosphatases into tartrate-resistant isoenzymes in vitro.

1. Chicken skeletal tartrate-sensitive (TsACP) and -resistant (TrACP) acid phosphatase isoenzymes could be separated from each other by carboxylmethyl-sepharose ion exchange chromatography. 2. Chicken skeletal TsACP showed a gradual time-dependent loss of sensitivity to tartrate inhibition when incubated at room temperature, but not at 4 degrees C. 3. The loss of sensitivity to tartrate inhibition was associated with an activation of the enzyme activity. 4. These changes were accompanied with a shift in the electrophoretic mobility of the enzyme activity from a large molecular sized form to a smaller molecular sized form that resembled the freshly prepared TrACP on the native acidic polyacrylamide electrophoresis gels, and on molecular sieve Superose-12 Fast Protein Liquid Chromatography. 5. Kinetic evaluations of the biochemical properties of the "converted" TsACP activity resembled the TrACP. 6. The apparent "conversion" was not unique to chicken TsACP, since similar "conversion" was observed with partially purified preparations of bovine bone matrix TsACP and of human osteoblastic TsACP. 7. Addition of several serine protease inhibitors did not prevent the "conversion". 8. These findings are consistent with the possibility that skeletal TsACPs are precursors of skeletal TrACPs.     

Heterogeneity of liver acid phosphatases in developing chick embryo

1. The development, localization and heterogeneity of acid phosphatase and a Zn(2+)-activated acid phosphatase in cellular fractions of developing chick liver were studied. 2. Acid phosphatase is distributed abundantly in the particulate and soluble fractions. The soluble fraction is rich in Zn(2+)-activated acid phosphatase, which attains its peak activity at about 15 days of incubation. 3. The particulate acid phosphatase activity is inhibited by fluoride but not by sodium l(+)-tartrate or cysteine. On the other hand, the soluble Zn(2+)-activated acid phosphatase activity is inhibited by sodium l(+)-tartrate and cysteine but not by fluoride. 4. The pH optimum of these two enzymes is similar at about 5.6. 5. The soluble Zn(2+)-activated acid phosphatase activity appears to be thermally stabilized by the treatment with Triton X-100 or bovine serum albumin.