Control of Aspartate Transcarbamylase activity by Norit-A adsorbable compounds during synchronous growth of Chlorella pyrenoidosa

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1964-08-05
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Virginia Tech
Abstract

The factors which regulate enzyme activity in the growing cell have been the subject of active research during the past decade and are generally considered under the heading of "metabolic control". At least three control parameters must be considered as regulating the active level of a given enzyme: control of enzyme synthesis at the gene level, control of enzyme activity by small molecule metabolites once the enzyme has been synthesized, and control of enzyme activity imposed by its structural orientation within the cell and/or its organelles (which would affect permeability of substrates, cofactors, etc.). It can be hypothesized that periodism in the intracellular level of a given metabolite must be accompanied by periodism in the active level of one or more of the enzymes responsible for the synthesis or breakdown of this metabolite.

Synchronized cultures of microorganisms afford a unique tool for studying periodic changes in the intracellular levels of metabolites during cell growth. The studies presented in this thesis were designed to elucidate the relationships between the activity of Aspartate Transcarbamylase and factors which affect and are affected by the activity of this enzyme in synchronized cultures of Chlorella pyrenoidosa.

Aspartate Transcarbamylase, the first enzyme in pyrimidine biosynthesis, has been reported to be controlled by a phenomenon known as " product-inhibition". This enzyme was located in the soluble supernatant of a 100,000 x g preparation of sonicated C. pyrenoidosa ceils. The pH optimum (9.2), temperature optimum (approx. 37°), and stability characteristics of this enzyme from this organism are reported.

Aspartate Transcarbamylase when measured during two consecutive synchronous growth cycles in C. pyrenoidosa under continuous illumination was found to increase at alternating exponential rates in each growth cycle. When the increase in Aspartate Transcarbamylase was expressed in its logarithmic form, a plot with linear segments was obtained, each segment having a different slope.

The rate of increase of Aspartate Transcarbamylase activity during early daughter cell development changes (increases) at the 5th h. The 5th through 9th h of cell growth, where Aspartate Transcarbamylase activity is increasing at an accelerated rate, corresponds to the premitotic and nuclear division stages in the cell. Correlated with this period of increased Aspartate Transcarbamylase activity (5th through 9th h) there is an increase in the rate of accumulation of RNA and DNA concurrent with increase in the rate of accumulation of intermediates in the acid-soluble, Norit-A adsorbable-P pool (which contains the nucleotide-P fraction). At. approximately the 9th h of cell growth there is a decrease in the rate of increase of Aspartate Transcarbamylase activity. Evidence is presented indicating that the depression of activity during this period is at least partly due to the presence of Norit-A adsorbable compound(s) present in the cell (pyrimidine nucleotides, the principal inhibitors of this enzyme, would be Norit-A adsorbable). At the 8th h of cell growth ( 1h prior to the period of depressed Aspartate Transcarbamylase activity) the acid-soluble, Norit-A adsorbable-P pool reaches a maximum value (as % of total cellular-P). These data together with inhibition studies with a variety of nucleoside mono-, di-, and tri-phosphates suggest that pyrimidine nucleotides may be factors regulating Aspartate Transcarbamylase activity during cellular development.

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