Substrate (chemistry)

About: Substrate (chemistry) is a research topic. Over the lifetime, 35902 publications have been published within this topic receiving 740722 citations. The topic is also known as: enzyme substrate.

Some properties of purified phospholipase D and especially the effect of amphipathic substances

Abstract: 1. The soluble phospholipase D of cabbage was purified by heat treatment, acetone precipitation and electrophoresis on a density gradient of aqueous glycerol. 2. The purified enzyme slowly attacked a lecithin suspension whereas ultrasonically treated lecithin was hydrolysed more rapidly. 3. Diethyl ether stimulated the hydrolysis of both the lecithin suspension and ultrasonically treated lecithin. 4. Ca(2+) was essential for the hydrolysis (optimum about 0.04m); it could not be replaced by Mg(2+) or cationic amphipathic substances. 5. The reaction had a sharp pH optimum at pH5.4, irrespective of the physical form of the lecithin substrate or the activator used. 6. Anionic amphipathic substances such as dodecyl sulphate, phosphatidic acid, triphosphoinositide and monocetyl phosphoric acid, were potent activators of the reaction: other acidic lipids were relatively inactive. 7. Cationic amphipathic substances inhibited the hydrolysis; however, they also reversed the inhibition caused by using an excess of anionic amphipathic substance as activator. 8. The activation produced by amphipathic substances could not be correlated with their effect on the zeta-potential or size of the substrate particles. 9. The addition of activating anionic amphipaths to lecithin induces the latter to adsorb enzyme from solution. In the absence of Ca(2+) the enzyme is denatured on the highly negatively charged surface, but in the presence of Ca(2+) (or Mg(2+)) it is protected from denaturation. It is suggested that this adsorption is an essential prerequisite for ready enzymic hydrolysis. 10. The hydrolysis of lecithin by the enzyme was strongly inhibited by protamine sulphate (0.1mg./ml.) and by choline and ethanolamine. 11. Ultrasonically treated phosphatidylethanolamine, or mixed particles of phosphatidylethanolamine plus dodecyl sulphate, were slowly attacked by the enzyme provided that Ca(2+) was present.

Potassium activated phosphatase from human red blood cells. The mechanism of potassium activation.

Abstract: 1. The kinetic behaviour of the K-activated phosphatase in human red blood cell membranes has been investigated. The concentration of Mg required to give optimal activation is independent of substrate and K concentration, suggesting that Mg combines with the enzyme at a site that is independent of and non-interacting with the substrate and K sites.2. The effects of K are competitively antagonized by Na. Ouabain in suitable concentrations selectively abolishes the activating effect of K.3. Comparison between the hydrolysis of acetylphosphate by intact red cells and by fragmented ghosts suggests that the active site for the substrate is only accessible at the internal surface of the cell membrane.4. The plot of the total rate of p-nitrophenylphosphate hydrolysis versus substrate concentration can be fitted at any K concentration by a rectangular hyperbola. The effect on the total rate of increasing K concentration is exerted mainly on the apparent affinity for the substrate, which increases about 5 times as K goes from 0 to 55 mM. There is also a much smaller increase in the maximum velocity (about 1.3 times) for the same range of K concentrations.5. If the difference between the activity in the absence and in the presence of K is plotted as a function of substrate concentration, the curves obtained are no longer hyperbolic but pass through a maximum and then tend to a lower value.6. This kinetic behaviour can be explained much more easily by assuming that a single enzyme is responsible for the hydrolysis of the substrate in the presence and in the absence of K. A simple kinetic model based on this assumption was developed and when experimentally determined constants were fitted into the equations that predict its behaviour a reasonably good agreement between theory and experiment was obtained.7. In the ;single enzyme' model inhibitors that selectively abolish the K-dependent activity would act by blocking the combination of the enzyme with K. A simple treatment based on this idea was developed for the case of Na and its predictions were fulfilled by the experimental results.8. In the ;single enzyme' model the K-coupled hydrolysis is always larger than the difference between the rates in the presence and in the absence of K, and when K concentration is non-limiting the K-coupled rate is equal to the total rate.

Enzyme additions as a tool to assess the potential bioavailability of organically bound nutrients

Abstract: Potential enzyme activities in soil and water samples are measured by addition of an excess amount of suitable substrate and subsequent determination of product release. If the approach is reversed and an excess of enzyme is added, substrate availability becomes rate-limiting and the maximum release of product indicates the availability of a given substrate in a sample. This approach has been used in a range of studies using phosphatase enzyme additions to soil, manure and sediment extracts, soil suspensions, and lake and sea water ( n = 41). Significantly fewer studies have used enzymes from the carbon, nitrogen and sulfur cycles ( n = 14). In this review, the methodological aspects of enzyme additions are discussed using examples from studies in which enzymes from the phosphorus cycle were used. A meta-analysis performed for various soil extracts and water samples revealed that the majority of studies (75th percentile) indicate availability of organic phosphorus to enzymatic hydrolysis of up to 60%, with crude phytase preparations showing the lowest substrate specificity and greatest release of phosphorus. Compared to addition of enzymes from the phosphorus cycle, lower substrate degradation was generally achieved by addition of enzymes from the carbon, nitrogen and sulfur cycles to soil suspensions and soil organic matter extracts. Enzyme additions can be a valuable tool in process research, provided all the necessary controls are included and assay conditions optimized to ensure that the reaction reaches completion. Recommendations for the development of a standard protocol are made.