Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

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It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca{sup 2+}. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca{sup 2+} is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca{sup 2+} , the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the ... continued below

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BISSELL, MINA J.; TOSI, ROBERTO & GORINI, LUIGI June 29, 1970.

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It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca{sup 2+}. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca{sup 2+} is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca{sup 2+} , the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca{sup 2+} and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca{sup 2+} protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca{sup 2+} addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope. The proteinase excreted by a Sarcina strain (Coccus P) is found only in cultures containing Ca{sup 2+} ions (1), a feature common to proteinases of other bacteria (4, 12, 18) and to other excreted enzymes (14). Among the nontoxic divalent cations, Ca{sup 2+} is rather specific in this effect. Other ions such as Mn{sup 2+} or Mg{sup 2+}, the latter being present in all media as an indispensible growth factor, are ineffective. Addition of Ca{sup 2+} to the proteolytically inactive supernatant fluid of a calcium- free culture does not result in the appearance of the missing enzyme activity. The early assumption that Ca{sup 2+} was needed for enzyme synthesis or excretion (1) was challenged when the observation was made (5) that Ca{sup 2+} and not Mn{sup 2}, Mg{sup 2+}, Sr{sup 2+}, or Ba{sup 2+} was needed for preventing irreversible loss of activity of several bacterial proteinases. In particular, in the case of the excreted proteinase of Coccus P, it was shown (17) that this irreversible inactivation is due to autodigestion occurring in the absence of Ca2 . An antiwetting agent, Ficoll, delays this autodigestion, suggesting that the function of Ca{sup 2+} is to stabilize an already active form of the enzyme molecule rather than to act as a constituent of the prosthetic group required for activity. It has also been observed that, when Coccus P is grown in a complex proteose peptone medium, the proteinase appears abruptly late in the growth of the culture. The sudden burst of activity was explained by demonstrating the presence of a zymogen which is activated autocatalytically (8). The late appearance of activity was accounted for when it was discovered that in minimal medium containing Ca{sup 2+}, Coccus P excreted the proteinase immediately at the onset of growth (9), but that addition of Casamino Acid hydrolysate delayed enzyme production for a length of time roughly proportional to the amount added (H. Ennis and L. Gorini, 1959, unpublished data). A similar amino acid effect was observed for other proteolytic bacteria (3, 13). It was assumed, therefore, that in the absence of amino acids an unrestricted proteinase production could be found. However, another deviation, from a constant relationship between amount of enzyme and amount of cells producing it, became evident by using minimal medium. The rate of accumulation of enzyme decreased gradually, long before exponential growth had slowed down (T. Heyman and L. Gorini, 1955, unpublished data). As yet, no explanation for this decline has been provided. In this paper, in addition to studying the role of Ca{sup 2+} in enzyme production, we also analyze the kinetics of enzyme appearance and accumulation in minimal medium. It is found that Coccus P does not require Ca{sup 2+} for proteinase synthesis or excretion but only in so far as it is essential for enzyme stability. It is further found that the factor responsible for the decline in enzyme accumulation, observed under conditions in which enzyme inactivation or autodigestion is prevented, is the proteolytic activity of the enzyme itself. Thus, in addition to synthesis, excretion, activation, and stability, a novel element plays a role in controlling enzyme accumulation in the culture fluid.

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  • Journal Name: Journal of Bacteriology; Journal Volume: 105; Journal Issue: 3; Related Information: Journal Publication Date: 3/1971

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  • Report No.: LBNL-4376E
  • Grant Number: DE-AC02-05CH11231
  • Grant Number: 5 ROI Al 02011-12
  • Office of Scientific & Technical Information Report Number: 1009842
  • Archival Resource Key: ark:/67531/metadc833284

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  • June 29, 1970

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  • May 19, 2016, 3:16 p.m.

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  • June 16, 2016, 12:46 p.m.

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BISSELL, MINA J.; TOSI, ROBERTO & GORINI, LUIGI. Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P), article, June 29, 1970; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc833284/: accessed June 23, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.