A thiol-activated aminopeptidase of the pituitary

Aminopeptidase B was purified from goat brain with a purification fold of ∼280 and a yield of 2.7%. The enzyme revealed a single band on both native acrylamide gel and SDS-PAGE thereby confirming apparent homogeneous preparation and its monomeric nature. The enzyme exhibited a molecular mass of 80.2 kDa and 79.7 kDa on Sephadex G-200 and SDS-PAGE respectively. The pH optimum was 7.4 and the enzyme was stable between pH 6.0 and 9.0. l-Arg-βNA was the most rapidly hydrolyzed substrate followed by Lys-βNA. The K_m value with Arg-βNA was found to be 0.1 mM. Metal chelating and –SH reactive agents strongly inhibited the enzyme activity. 1,10-Phenanthroline exhibited mixed type of inhibition with a K_i of 5 × 10⁻⁵ M. The enzyme was highly sensitive to urea. Metal ions like Ni²⁺, Cd²⁺, Fe²⁺and Hg²⁺ inhibited the enzyme, whereas Co²⁺, Zn²⁺, Mn²⁺and Sn²⁺ slightly activated the enzyme.

Exopeptidases identified as dipeptidyl peptidases II and III were found in the bovine, human normal and human cataractous lenses. Arylaminopeptidase activities identified in extracts of these tissues were able to hydrolyze phenylalanine, leucine, methionine, lysine and alanine arylamidase substrates. Lysosomal DAP II and the cytosol DAP III were recognized by their marked preferences for the release of Lys-Ala from Lys-Ala-2-NNap (at pH 5·5) and Arg-Arg from Arg-Arg-2-NNap (at pH 9·0), respectively. DAP III in human lens extracts exhibited a pH optimum of 9·5 at 37°C. The K_m for the DAP III substrate was estimated to be 4·64 × 10⁻⁵ m for normal lens extracts, 2·74 × 10⁻⁵ m for human cataractous lens extracts, and 2·83 × 10⁻⁵ m for bovine lens extracts. Lens DAP III was strongly inhibited by PCMS, EDTA, and to a lesser degree by puromycin and DFP. Arylaminopeptidase activities (from human and bovine lenses) assayed on Leu-2-NNap were strongly inhibited by PCMS and puromycin. Negligible effects were obtained with EDTA and DFP. Only traces of leucyl aminopeptidase (assayed on leucinamide) could be detected in human lenses; however, aminopeptidase activities measured at pH 7·0 on the β-naphthylamides of leucine, methionine, lysine, phenylalanine and alanine were plentiful in both human and bovine lenses, and served to indicate the possible presence of other aminopeptidases such as alanine aminopeptidase and aminopeptidase B. Of particular interest was the finding that the specific activities of Met-, Lys-, and Ala-2-NNap were 2–5-fold higher in cataractous human lenses compared to normal lenses.

Greenberg's fluorimetric assay for arylamidase N activity (1) has been standardized and modified for determination of the activity in small freezedried neural lobe tissue samples (0.2–1.0 μg).

Water extracts from bovine hypophysis and hypothalamus did not hydrolyze casein in neutral and alkaline pH region, but did split azocoll and the synthetic hexapeptide Cbz–Gly–Pro–Ala–Gly–Pro–Gly–OCH₃ at the bond Ala–Gly. A partially purified enzyme from the anterior lobe of hypophysis also split a series of analogues of the hexapeptide studied, but did not reduce the viscosity of rat skin procollagen solution. The preliminary estimation of the molecular weight of the enzyme gave the value of 50 000. Its pH optimum was 7–9. The ability of the enzyme to hydrolyze N- and C-substituted hexapeptides, which are known to be the specific substrates for collagenase, suggests the existence of an endopeptidase with collagenase activity in the tissues of hypophysis and hypothalamus.

Human liver arylamidase was purified 2257-fold by a slightly modified procedure. The enzyme preparations were homogeneous in the analytical ultracentifuge and produced a single protein band on disc electrophoresis.

The molecular weight for the native enzyme calculated from sedimentation, diffusion, and gel filtration data, was 234 600 ± 9300. The sedimentation coefficient was 10.1 · 10⁻¹³ sec and the diffusion coefficient was 4.02 · 10⁻⁷ cm²/sec.

The enzyme also gave only one component when studied in the analytical ultracentrifuge in 6.0 M guanidine−0.1 M β-mercaptoethanol. Agarose chromatography of the enzyme in this same medium produced data from which a reduced molecular weight of 38 000 ± 2200 was obtained. In 8.0 M urea only partial dissociation occurred. These data suggest that the enzyme consists of six subunits of equal molecular weight.