Purification and characterization of guinea-pig epidermal acid phosphatase

Guinea-pig epidermal acid phosphatase has been purified approximately 120-fold by a procedure including acid treatment, CM-cellulose and DEAE-cellulose chromatography, and gel filtration on Sephadex G-100. The enzyme had a pH optimum at 5-0 and the optimal temperature for activity was approximately 50 degrees C. The enzyme was not activated by divalent cations or 2-mercaptoethanol, but it was inhibited by p-chloromercuribenzoate and by fluoride. The km value for p-nitrophenyl phosphate was 1-31x10-4 M, the molecular weight was about 73,000 as determined by Sephadex G-100 gel filtration and the isoelectric point was 6.1. The enzyme hydrolyzed deoxyribonucleoside monophosphates to deoxyribonucleosides.     

Enzymological studies of epidermal acid phosphatase.

Acid phosphatase of newborn rat epidermis was purified about 270-fold by a procedure including acid treatment, CM-cellulose, DEAE-cellulose chromatography, and gel filtration on Sephadex G-100. The purified enzyme had a pH optimum of 5.0 and an optimal temperature of 40°C. The enzyme was not activated by divalent cations, ethylenediaminetetraacetic acid or 2-mercaptoethanol, and it was inhibited by p-chloromercuribenzoate. The inhibition pattern by fluoride and by L(+) tartrate was competitive. The Km value for p-nitrophenyl phosphate was 3.84×10^?5 M, and the molecular weight was about 73, 000. The enzyme appeared to be a nonspecific acid phosphatase. Intracellular localization of acid phosphatase in the epidermis was discussed.     

Tyrosinase and acid phosphatase activities in melanocytes from avian albinos

Two forms of cutaneous albinism in the chicken were investigated for the presence and distribution of tyrosinase and acid phosphatase in melanocytes in situ and in culture. In sex-linked recessive tyrosinase-positive albinism, sal, melanocytes in regenerating feathers and neural tube-derived cultures contained morphologically normal and abnormal premelanosomes. Tyrosinase was localized primarily to the abnormal premelanosomes and probably not to the normal ones. The cells possessed, in addition, vacuoles with membranous inclusions, located in the dendrites, and capped by dopa-positive vesicles (capping vesicles). Acid phosphatase colocalized with tyrosinase in the abnormal premelanosomes and capping vesicles. Tyrosinase activity in extracts of cultured sal melanocytes equalled that of e+ control melanocytes. A tyrosinase antiserum, raised against hamster tyrosinase (Pomerantz), precipitated 2 proteins, 68 kD and 82 kD, which had a precursor-product relationship. The amount of immunoprecipitate was the same in sal and control extracts, but in sal extracts the lower-molecular-weight protein was twice as abundant as the higher-molecular-weight protein. Melanocytes in regenerating feathers from an autosomal recessive, tyrosinase-negative albino, ca, also contained morphologically normal and abnormal premelanosomes. In culture, ca melanocytes had no formal premelanosomes but only dopa-negative multivesicular bodies with wispy filamentous material. Tyrosinase activity and immunoprecipitable tyrosinase were absent. These results suggest that: the tyrosinase-positive albino, sal, has an aberration in both its tyrosinase and acid phosphatase profiles and the tyrosinase-negative albino, ca, lacks functionally and antigenically normal tyrosinase.     

Properties of acid phosphatase in human stratum corneum

Sheets of stratum corneum were prepared by a trypsinization procedure from human skin samples, homogenized with a freeze press and then fractionated into a soluble fraction and a sediment by centrifugation at 50,000 g. Acid phosphatase (AcP) activity was found in both fractions but the bulk of the activity was detected in the supernatant. Highest activities were observed after treatment with Triton X-100. The bulk of the AcP activity remained bound to the pellet, if suspension and fractionation of the homogenized stratum corneum were performed in acetate buffer in the range between pH 4.0-5.0, probably due to ionic or hydrophobic interactions. AcP activity was totally lost if homogenates or fractions were stored frozen at -20 degrees C in buffers with pH values lower than 4.0. Triton X-100 extracts from whole skin, epidermis, stratum corneum, cultured skin fibroblasts and leukocytes were compared by isoelectric focusing. Extracts from whole skin, epidermis and stratum corneum yielded almost identical patterns with one main AcP activity band at pI of 5.65, whereas a second pronounced band from whole skin behaved similarly to one band from cultured skin fibroblasts and leukocytes (pI 6.1). The prominent band from extracts of stratum corneum and epidermis was not observed in extracts of skin fibroblasts and leukocytes. Hence, we conclude that stratum corneum and epidermis contain a tissue-specific AcP.     

Citrate and acid phosphatase in the ejaculate in prostatic carcinoma and adenoma]

The simultaneous determination of acid phosphatase and citrate concentration in the seminal fluid obtained from 16 patients with prostatic adenoma showed normal values, 6 patiients with prostatic carcinoma (cytologically and histologically verified) however extremely low values. The difference between persons with prostatic cancer and those with adenoma became particulary obvious with both experimental results evaluated in the way of a twodimensional diagram. This clear separation of both clusters by the simultaneouse estimation of both biochemical parameters may possible get useful diagnostic significance.     

Biochemical studies on a novel vanadate- and molybdate-sensitive acid phosphatase from human epidermis

A novel vanadate- and molybdate-sensitive human skin epidermal acid phosphatase was purified and characterized. The enzyme was extracted from epidermal sheets with a 0.1% Triton X-100 solution buffered at pH 7.0. The purification procedure consisted of molecular permeation chromatography on Sephadex G-200 followed by chromatography on hydroxylapatite using an ammonium sulfate gradient. The molecular weight of the enzyme was 82,000 and the isoelectric point was at pH 5.6. At the optimum pH (5.1) the enzyme hydrolyzed most rapidly 1-naphthyl phosphate (Km = 0.28 mM) and 4-nitrophenyl phosphate (Km = 0.28 mM). In general, the best substrates had an aromatic leaving group. Fluoride (Ki = 39 microM; noncompetitive) and phosphate (competitive) inhibited by binding to different binding sites of the enzyme. The most potent inhibitors were vanadate (Ki = 1.9 X 10(-6)M), tungstate (Ki = 1.4 X 10(-7)M), and molybdate (Ki = 2.0 X 10(-9)M). Chemical modification and kinetic experiments suggested that the activity of the enzyme is based on imidazole, tyrosyl, and carboxyl groups. Benzoyl peroxide was a relatively potent inhibitor (Ki = 5.0 X 10(-5)M; noncompetitive). This enzyme resembled the prostatic acid phosphatase with regard to substrate specificity, inhibition characteristics, and functional groups.     

Changes in epidermal acid phosphatase levels in response to chemical irritation

Topical sodium laurate produced little or no immediate change in the amount of rat epidermal acid phosphatase but the levels of this enzyme progressively increased to reach a maximum at about 3 days. This coincided with acanthosis and thickening of the stratum granulosum, indicating epidermal repair. The increase depended on protein synthesis as it could be blocked by treatment with cycloheximide. This inhibitory effect was associated histologically with loss of the stratum granulosum, supporting the belief that the expression of acid phosphatase is closely linked with this layer during normal keratinization.     

The alkaline phosphatase anti-alkaline phosphatase technique in dermatopathology

The APAAP technique is an unlabelled antibody bridge technique which can be used on either frozen or paraffinembedded sections. One applies first a monoclonal antibody, then a polyclonal bridge antibody, and finally a soluble complex of alkaline phosphatase and monoclonal mouse anti-alkaline phosphatase. Subsequently, the enzyme label is developed with a naphthol salt and new fuchsin as a dye. This technique was used in our laboratory on frozen and/or paraffin embedded sections by using 15 different monoclonal antibodies, which are commercially available. The reaction product was bright red and could easily be distinguished from the brown color of melanin, which makes the APAAP technique particularly suitable for dermatopathology.