A specific inhibitory protein to a restriction enzyme from Saccharomyces cerevisiae


Mukaram Shikara*

A specific protein inhibitor for the restriction enzyme (SacC1) has been purified from Saccharomyces cerevisiae approximately 21,000 fold and its inhibitory properties have been characterized. The isoelectric points (pI) of SacCI and its inhibitor are 9.0 and 5.22, respectively. The molecular weight of SacC1, the inhibitor and SacC1-inhibitor complex were estimated by gel filtration on a Sephadex G-100 column to be 64,000, 32,000 and 85,000, respectively. The inhibitor protein inhibits SacC1 catalytic activities efficiently, but has no effect on other restriction enzymes tested. Inhibition does not occur unless SacC1 enzyme is exposed to the inhibitor protein prior to the reaction of the enzyme with DNA. The inhibitory activity is independent of temperature. The inhibition increased linearly with the addition of inhibitor to various amounts of SacC1, up to 85% inhibition. The slope of inhibition was constant irrespective of the initial amount of SacC1 and Ki value of 3.45 x 10-12 was obtained. The inhibitor interacts strongly with SacC1 and this interaction could increase the stability of the complex, possibly manifesting itself as SacC1 decreases in the dissociation rate due to the electrostatic attraction between the two groups or the stability may increase by potentially stronger electrostatic interaction. The conformational specificity between SacC1 and its inhibitor seems to be essential for their interaction. The extremely strong affinity of the inhibitor to SacC1 is remarkable and stronger than the affinity of several restriction enzymes

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