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The capability of guinea pig epidermal extracts to hydrolyze deoxyribonucleic acid has been studied. The results of investigation by gel filtration on Sephadex G-75 column and by viscometry on the mode of hydrolysis of deoxyribonucleic acid by epidermal extracts revealed that deoxyribonucleic acid was degraded by both endonuclease and exonuclease activities. The exhaustive digestion by epidermal extracts yielded the complete degradation of deoxyribonucleic acid to mononucleosides or further metabolites. The enzyme systems involved in the deoxyribonucleic acid degradation include at least an endonuclease, an exonuclease and a phosphatase.

Guinea pig epidermal DNAase I was purified from an epidermal extract by a procedure including DEAE-cellulose chromatography, Sephadex G-100 gel filtration and Con A-Sepharose affinity chromatography. The purified enzyme contained no detectable activities of acid DNAase, alkaline RNAase, phosphodiesterase or acid or alkaline phosphatase, but was contaminated with acid RNAase activity. The molecular weight of the enzyme was estimated to be 33 000 by sucrose density gradient centrifugation and Sephadex G-100 gel filtration. Its isoelectric point is 5.2 +/- 0.1. The enzyme requires divalent cations and exhibits two pH optima that are dependent on divalent cations: in the presence of Mn2+, the optimum pH is about 7.5 in 50 mM Tris-HCl buffer and in the presence of Mn2+, the pH is 6.4 in 50 mM cacodylate-HCl buffer. The enzyme hydrolyzes native DNA about 6-times faster than denatured DNA, producing 5'-phosphoryl and 3'-hydroxyl terminated oligonucleotides with an average chain length of about eight nucleotides, and converts double-stranded and circular DNA to relaxed and linear forms. The enzyme is inhibited by G-actin and antiserum against bovine pancreatic DNAase A. Thus this enzyme is classified as DNAase I.

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.

Isoelectric focusing and concanavalin A-Sepharose chromatography were used to study the multiple forms and glycoprotein natures of so-called lysosomal hydrolases from psoriatic scales. Acid phosphatase appeared as 5 different forms with pI values of 6.5, 6.1, 5.8, 5.6 and 5.45. Seven isoenzymes of alpha-fucosidase were identified with pI values of 6.4, 6.2, 5.9, 5.75, 5.65, 5.4 and 5.2. Acid alpha-mannosidase activity appeared as one peak with pI value of 6.75 and a weak activity of neutral alpha-mannosidase was present with pI value of 6.7. Incubation of the extract with neuraminidase increased their pI values of acid phosphatase, alpha-fucosidase and alpha-mannosidase to the more basic forms. This finding suggests that epidermal acid phosphatase, alpha-fucosidase and alpha-mannosidase have some N-acetylneuraminic acid residues. In addition concanavalin A-Sepharose column chromatography was also performed to confirm the glycoprotein nature of acid phosphatase. This enzyme was bound to the column and not released from the column even with the treatment of 0.5 M NaCl, but the enzyme was eluted from the column with the treatment of alpha-methyl-D-glucoside.

An acid deoxyribonuclease of guinea pig epidermis hydrolyzes chromatin DNA as well as histone-free DNA. The enzyme degrades approximately 50% of chromatin DNA, while it degrades 90% of histone-free DNA. Guinea pig epidermal extract contains proteolytic activity for histone or chromatin with a pH optimum at around 8.0.

Deoxyribonuclease was isolated from cow snout epidermis and purified approximately 200-fold in specific activity. The enzyme has a pH optimum of 5.5 and a temperature optimum at 50° C. No physiologic cations were required for enzyme activity. Activity was strongly inhibited by zinc sulfate and ammonium sulfate. It was found that the enzyme hydrolyzed native DNA more efficiently than heat denatured DNA. Because the average chain length of the limit digest was approximately 3, the isolated DNase was considered to be an endonuclease acting at many places along the length of the DNA polymer chain. The physiologic role of DNase in nuclear decomposition during epidermal keratinization is discussed.

Two chromatographically and enzymatically distinct forms of acid phosphatase have been separated by gel filtration from an extract of guinea-pig epidermis. These enzymes, termed APase 1 and APase 2 , had pH optima for p -nitrophenyl phosphate hydrolysis around 5.0. APase 1 was inhibited by fluoride, L(+) tartrate, and lead nitrate, but was insensitive to formaldehyde and glutaraldehyde. In contrast, Apase 2 was insensitive to fluoride, L(+) tartrate, and lead nitrate, but was sensitive to formaldehyde and glutaraldehyde. K m values for APase 1 and APase 2 with p -nitrophenyl phosphate as substrate were 0.09mM and 0.125 mM, respectively.

Two forms of alpha-fucosidase from newborn rat epidermis were separated by gel filtration on Sephadex G-150. These enzymes termed, fucosidase I which was eluted in the void volume and fucosidase II (molecular weight approximately 50,000). Both enzymes had pH optima for 4-methylumbelliferyl-alpha-L-fucoside hydrolysis between 5.5-6.5. Km values for fucosidase I and II with the same substrate were 3.7 X 10(-5) and 5.4 X 10(-5) M, respectively. Three different forms of new born rat epidermal alpha-fucosidase were separated by isoelectric focusing. Evidence is present which indicates that the electrophoretic heterogeneity of alpha-fucosidase is due in part to the binding of sialic acid to the primary gene product.

Two forms of beta-galactosidase from newborn rat epidermis could be separated by DEAE-cellulose chromatography. Both enzymes showed similar enzymic properties. They had a pH optimum around 3.5--4.5 and the optimal temperature of these enzymes was approximately 60 degrees C. They were not affected by divalent cations, ethylenediaminetetraacetic acid(EDTA) and 2-mercaptoethanol(2-ME), while rho-chloromercuribenzoic acid (PCMB) was a strong inhibitor for each enzyme. These enzymes showed the same Km value (1.25 x 10(-4) M) towards 4-methylumbelliferyl-beta-D-galactoside. However they had different isoelectric points at pH 6.3 and 9.0, respectively. Six different forms of beta-galactosidase activity were found by using isoelectric focusing. When the crude extract was incubated with neuraminidase before electrofocusing, the acidic forms of the enzyme were largely lost and converted to more basic forms without loss of the total activity. This finding suggests the glycoprotein nature of newborn rat epidermal beta-galactosidase.