PERIPLASMIC AMINOPEPTIDASE AND ALKALINE-PHOSPHATASE ACTIVITIES IN A MARINE BACTERIUM - IMPLICATIONS FOR SUBSTRATE PROCESSING IN THE SEA

The occurrence of periplasmic aminopeptidase and alkaline phosphatase (APase) was studied in a Gram-negative bacterium (designated S8) isolated from the surface waters off the Southern California coast. We tested the hypothesis that processing of polymeric substrates (e.g. protein) by marine bacteria involves ectohydrolases both on the cell surface and in the periplasmic space but small substrates (e.g. sugar phosphates) are hydrolytically processed only in the periplasm. Optimum conditions for cold osmotic shock were established to maximize protein release (14.3 +/- 2.2 % of the total cellular protein) without significant release of cytoplasmic protein (using glucose-6-phosphate dehydrogenase as cytoplasmic indicator). Only 4.2 +/- 4.0 % aminopeptidase but 63.4 +/- 4.8 % APase was shockable. An additional 17 % APase was released by a second shock. Although not shockable, the majority of aminopeptidase (67 %) was periplasmic [not accessible to bovine serum albumin (BSA)]; 33 % was considered surface bound (accessible to BSA). The cellular distribution of aminopeptidase activity is consistent with the model that cell surface aminopeptidase generate oligomers which diffuse into the periplasm where they are monomerized by the high aminopeptidase activity in an environment where the monomers may interact with substrate binding protein or directly with the permeases. While most APase was periplasmic we were unable to determine whether any APase was also present on the cell surface. In S8 the fluorogenic substrates (leucine AMC and MUF-P) did not measure the cytoplasmic enzymes but did measure periplasmic activities. If these results can be extrapolated to natural marine assemblages, then ectoenzyme measurements using the fluorogenic substrates represent the sum of cell surface and periplasmic (but not cytoplasmic) pools of the enzymes. Since ectohydrolase distribution may be important in substrate processing and bacteria-organic matter coupling, future environmental studies of ectoenzymes should distinguish between the 'surface ectoenzymes' and 'periplasmic ectoenzymes'.