
%0 Journal Article
%A Ratcliffe, Catherine 
%A Sanders, RL 
%A Ludmila Tittel, C 
%A O’Brien, RW 
%T Amylase and Protease Secretion by the Marine Bacterium Vibrio gazogenes
%J Australian Journal of Biological Sciences
%D 1982/08/01
%V 35
%N 4
%P 457-467
%K 
%R https://doi.org/10.1071/BI9820457
%U https://www.publish.csiro.au/paper/BI9820457 
%X V. gazogenes secreted an amylase throughout the logarithmic phase of growth when starch or maltose was the carbon source for growth. The enzyme was apparently not constitutive and was repressed by glucose. The amylase was of the oc-type and had an optimum pH of 6&#xb7;5. Protease secretion by V. gazogenes occurred when the organism was grown in a defined medium containing 0&#xb7;005 % (w/v) yeast extract. The activity of the enzyme was increased 40-fold when the organism was grown in a medium containing peptone or Casamino Acids. Enzyme secretion started in the late logarithmic phase of growth and ceased when the cells entered the stationary phase of growth and was not repressed by glucose. The protease was neither induced nor repressed by glutamic acid, aspartic acid, alanine, serine, arginine, valine, threonine, lysine, leucine, proline or oc-ketoglutarate. Tryptophan delayed, but did not inhibit, protease secretion. Both the amylase and the protease were truly extracellular and were not released as a result of cell lysis. Phenylmethylsulfonyl fluoride and N-oc-p-tosyl-L-Iysine chloromethyl ketone hydrochloride, L-l-tosylamide-2-phenylethyl chloromethyl ketone, leupeptin, anti pain and chymostatin did not markedly inhibit the protease, indicating that it is not a serine protease nor similar to trypsin, chymotrypsin, papain or cathepsin B. Inhibitors of sulfhydryl enzymes were also without effect on protease activity, but 1,1O-phenanthroline, ethylenediaminetetraacetate and ethyleneglycol-bis-(p-aminoethyl ether) N,N'-tetraacetate almost completely inhibited the protease, indicating that it requires a divalent metal ion for activity. After dialysis against water, both amylase and protease lost over 90% of their activity. The amylase was almost completely reactivated by 15 mM Cl-, Be or 1-, but not by Ca 2+ or Mg2+. Protease was fully reactivated by about 30 mM Ca2+ or Mg2+ (at 70 ruM concentration these ions stimulated activity to about 140% of the rate of the undialysed enzyme). Mn2+ and Co2+ partially restored protease activity. Chloramphenicol, at concentrations that did not affect RNA synthesis, completely inhibited amylase and protease secretion, showing that secretion of both enzymes was a de novo process. When rifampin or actinomycin D, at concentrations that completely and rapidly inhibited cellular RNA synthesis, was added to cultures actively secreting amylase or protease, there was no inhibition of secretion for periods of 6-20 min, indicating the presence of a pool of mRNA specific for each enzyme. The protease was not affected by trypsin or oc-chymotrypsin during the secretion process, indicating that the enzyme had taken up its tertiary conformation before emergence from the cell membrane. 
