Enzyme-forming System Research Articles

Age-dependent modifications of lipogenic enzymes

When 1-, 2- and 9-month-old rats previously adapted to a commercial stock diet were fed a fat-free diet (induction) and also when the process was reversed (repression), the turnovers of lipogenic enzymes in liver were measured by following time courses for the change of the enzyme activities. The magnitudes of the induction of glucose-6-phosphate dehydrogenase, malic enzyme and acetyl-CoA carboxylase were very high in 1-month-old rats and then decreased with aging. In the induction phase, the rates of synthesis of the enzymes were markedly decreased with increasing age as compared with the rate constants of degradation. The age-dependent alterations were not shown so much in the repression phase as in the induction phase. It is suggested that the age-dependent impairment in induction may be due to some alterations in the enzyme-forming system.

Immunochemical studies of serine dehydratase and ornithine aminotransferase regulation in rat liver in vivo.

Previous studies of serine dehydratase (EC 4.2.1.13) and ornithine aminotransferase (EC 2.6.1.13) adaptation in rat liver showed that in rats on a high protein diet, glucocorticoid administration increased serine dehydratase activity while simultaneously reducing the activity of ornithine aminotransferase. The present study examines the role of enzyme synthesis in the expression of these and other dissimilar adaptive characteristics of the two enzymes. Both enzymes were purified to crystallinity and used to prepare specific antibodies. Changes in the rate of synthesis of each enzyme during adaptation were then measured immunochemically. In rats fed ad libitum, the synthetic rates for both enzymes exhibited circadian rhythm, although enzyme levels remained relatively constant. The circadian cycle for ornithine aminotransferase synthesis was in phase with the cycles for body weight and relative liver weight (maxima at 9 a.m., minima at 9 p.m.) but was approximately 12 hours out of phase with the cycle for serine dehydratase synthesis. 9alpha-Fluoro-11beta, 21-dihydroxy-16alpha, 17alpha-isopted at 9 a.m., increased serine dehydratase synthesis and simultaneously decreased the synthesis of ornithine aminotransferase. When triamcinolone was injected at 9 p.m., however, serine dehydratase synthesis was not stimulated, although the reduction of ornithine aminotransferase synthesis was still produced. These results suggest that: (a) circadian cycling of synthesis may be a general phenomenon in enzyme regulation even though for enzymes with relatively long half-lives, such cycling may not be reflected as fluctuations in enzyme levels; (b) such circadian rhythmicity may also involve cyclic changes in the responsiveness of the enzyme-forming system to regulatory stimuli; (c) whereas the adaptive behavior of serine dehydratase typifies that of amino acid-catabolizing enzymes in general, the responses of ornithine aminotransferase denote a functional association of this enzyme with anabolic processes. On this basis, the possibility that ornithine aminotransferase plays a pivotal role in the regulation of urea cycle activity and nitrogen balance is discussed.

Kinetics of hydrolase production by microorganisms

ABSTRACT The enzyme-forming systems (EFSs) concerned with the amylase of Bacillus subtilis, and the glucamylase and acid protease of Aspergillus niger, have been shown to be highly stable in the cultural conditions applied owing to the low decay rate of mRNAs specific for these respective enzymes. The quantity per cell of mRNAs for any of these enzymes is regarded as limiting the specific rate of enzyme production in the conditions used for enzyme production. Investigations on the repression, derepression and the preferential synthesis of these hydrolases have led to simple hypothetical relationships.

Effects of environmental agents at the level of enzyme-forming systems

Effects of environmental agents at the level of enzyme-forming systems

Regulation of gluconeogenesis and glycolysis: Studies of mechanisms controlling enzyme activity

Various regulatory mechanisms functioning at the enzyme activity and enzyme biosynthetic level in carbohydrate metabolism were explored. The regulatory influences operate on receptor sites on the enzyme molecules and on the enzyme-forming systems. These enzymatic control mechanisms were examined as the basis for rapid and chronic regulation of gluconeogenesis and glycolysis by hormones and regulatory signal molecules. This work presented the effects of NADH, free fatty acids, and other compounds on strategic enzymes of liver carbohydrate metabolism. The selective action of free fatty acids was characterized in terms of inhibition of key enzymes of glycolysis, and the direct oxidative pathway, and of certain enzymes in the Krebs cycle. Since liver pyruvate kinase is pivotal in the regulation of gluconeogenesis and glycolysis, detailed attention was given to the effects of fructose 1,6-diphosphate, ATP, ADP, NADH, magnesium, free fatty acids, and other regulatory molecules on this enzyme.

Industrial Fermentations and Their Relation to Regulatory Mechanisms

This chapter discusses the industrial fermentations and their relation to regulatory mechanisms. The success of an industrial fermentation does not depend on addition of precursors. Rather, the effects of regulatory mechanisms such as feedback inhibition and repression are of extreme importance. Modification of feedback effects without destroying the metabolic activities of the cell has become a major activity of the fermentation microbiologist. Such alteration in living cells can be done by decreasing the concentration of the end product, and modifying the sensitive enzyme or enzyme-forming system. Regulatory controls probably affect antibiotic production as well. The inhibition of penicillin formation by lysine and the stimulation of biosynthesis of cephalosporin C by methionine are apparently the results of normal feedback effects of these amino acids on their own biosynthesis. Knowledge of control mechanisms can be expected to stimulate effort towards the industrial production of microbial enzymes and nucleic acids. Constitutive mutants have already been obtained, producing 25% of their protein as a single enzyme. The chapter also briefly discusses the synthesis of macromolecules.

EFFECT OF NUTRITIONAL STATE ON HORMONAL REGULATION OF LIVER ENZYMES

The ability of enzyme-forming systems to meet the challenge of starvation and produce vital metabolites for maintaining homeostasis was studied. The preferential maintenance of hepatic gluconeogenic enzymes in starvation can be blocked by hypophysectomy and also by inhibitors of protein synthesis, actinomycin and ethionine. Starvation, cortisone, and triamcinolone were capable of inducing a pronounced increase in the incorporation of orotate into hepatic RNA. Actinomycin administration blocked the starvation- and steroid-induced rise in RNA specific activity. The glucocorticoid hormone induced increase in enzyme biosynthesis and the rise in free amino acid level were rapidly blocked by actinomycin and ethionine and were affected also by starvation.In the adaptation of the organism to acute starvation the hepatic enzyme-forming systems are regulated by the interplay of two hormones to produce a preferential maintenance of key gluconeogenic enzymes. Insulin, a suppressor of biosynthesis of gluconeogenic enzymes, is known to decrease in amount during starvation. Under the same circumstances glucocorticoid hormones, which are inducers of the biosynthesis of gluconeogenic enzymes, are present and thought to be responsible for the preferential maintenance of key gluconeogenic enzyme activities, thus ensuring adequate blood glucose levels in starvation.

CONTROL OF GLUTAMINE SYNTHETASE IN THE EMBRYONIC RETINA IN VITRO.

Glutamine synthetase activity in the neural retina of the chick embryo increases sharply during terminal differentiation of this tissue. This characteristic increase can be reproduced in cultures of retinal tissue fragments from late embryos. A similarly sharp increase can be elicited precociously in younger retina by culturing in medium with adult serum. Both the precociously elicited and the later increase in enzyme activity require continuous protein synthesis; both can be suppressed during the first 24 hours of culture by blocking RNA synthesis or by removing the adult serum. Subsequently, the increase in enzyme activity becomes progressively less dependent on RNA synthesis and on the continuous presence of adult serum. This transition is attained more rapidly in the older retina. The data suggest a progressive stabilization of the enzyme-forming system during differentiation.

SUBSTRATE AND HORMONAL INTERACTIONS IN THE REGULATION OF ENZYME LEVELS IN RAT HEPATOMAS.

Thus, of four enzymes studied in rat hepatomas, corticoids have been shown to affect their enzyme-forming systems minimally (TPO and G6PD), variably (TD) or maximally (TAKG). These are at present aberrations in control mechanisms, the control of protein synthesis, studied only at the physiological and cellular level. Steps are now being taken in this laboratory (17) to understand the molecular changes present in the hepatoma cell which are responsible for the observed metabolic defects.

Temperature-sensitive formic hydrogenlyase in a psychrophilic bacterium.

Upadhyay, J. (Washington State University, Pullman) and J. L. Stokes. Temperature-sensitive formic hydrogenlyase in a psychrophilic bacterium. J. Bacteriol. 85:177-185. 1963.-A temperature-sensitive formic hydrogenlyase has been found in a psychrophilic bacterium, strain 82. The enzyme was most active at 30 C, but completely inactive at 45 C. In contrast, formic hydrogenlyase in mesophilic Escherichia coli was most active at 45 C, and completely inactivated only at 70 C. The hydrogenlyase-forming system in psychrophile strain 82 also was unusually heat-sensitive. It was inactivated at temperatures above 20 C; whereas, in mesophilic E. coli, Proteus vulgaris, and Salmonella oranienburg, hydrogenlyase was formed even at 45 C. This heat sensitivity of the enzyme-forming system accounted fully for the inability of strain 82 to produce gas from carbohydrates above 20 C. Psychrophile hydrogenlyase resembled the mesophilic variety in several respects. It was most active at pH 5.0. It was not formed by cells grown aerobically. It could be induced in resting-cell suspensions by use of glucose, formate, and casein hydrolysate as supplements, although several times as much glucose was required as for enzyme induction in strains of E. coli and Salmonella. The enzyme was formed best at pH 7.5, which was somewhat higher than the pH of 6 to 7 for mesophile hydrogenlyase, and by cells taken from the beginning of the stationary phase of growth.

The effect of ultraviolet light and x rays on an enzyme-forming system.

Ultraviolet irradiation of E. coli cells was found to inhibit the induced synthesis of beta -galactosidase and the inhibition was reversed by photoreactivation. Examination of crude extracts of such cells led to the discovery of a particle-bound fraction of beta -galactosidase. The particlebound enzyme occurs only in the presence of inducer. When inducer is removed, the enzyme leaves the particle and becomes associated with the soluble pool. These observations led to the discovery of a cell-free system that can synthesize the enzyme. The system consists of particles and supernatant, both of which must be prepared from pre-induced cells. The system requires the presence of inducer, a source of energy, amino acids, and the nucleoside di- and triphosphates. Enzyme synthesis is inhibited by chloramphenicol, glucose, ribonuclease and deoxyribonuclease. Ultraviolet treatment of the supernatant yielded an inactive preparation that could be made active by addition of DNA from untreated supernatant. Similarly x irradiation of cells destroyed induced enzyme synthesis and cell-free preparations made from x-irradiated cells were inactive. Such inactive preparations could synthesize enzyme when supplemented with DNA. Only DNA's prepared from cells containing the gene for beta -galactosidase are effective. (auth)

The initial kinetics of enzyme induction

Kinetics of induction of several enzymes of Escherichia coli have been investigated under conditions where non-specific nutrient effects and permeability mechanisms are not important. Measurements made over time intervals of a few minutes permitted detection of initial events brought about by addition or removal of inducers or inhibitors. With each enzyme a lag of about 3 min between addition of inducer and appearance of enzyme at 37° was noted. This lag was dependent on temperature and independent of inducer concentration, in contrast to an inhibitor which showed the opposite behavior. The induction lag, therefore, does not represent the time required for penetration of the inducer. Studies with inhibitors suggest that some metabolism involving the synthesis of a ribonucleic acid is required before enzyme synthesis can proceed. The formation of enzyme ceased about 5 min after inducer was removed or after glucose was supplied to the culture, suggesting that the enzyme-forming system is unstable. A model for the enzyme forming apparatus of the cell is suggested which is consistent with the kinetic data and with other information regarding induction. A repressor prevents function of the genetic material of the bacteria. The inducer is thought to prevent the repressor from acting. This freed genetic material forms a special, unstable ribonucleic acid which interacts with the ribosomes to provide an active template for enzyme synthesis. The time required for enzyme formation to commence is attributed principally to the interval during which the repressor is lost and the unstable ribonucleic acid is formed. A possible similarity between repressor and unstable ribonucleic acid is suggested. Instability appears to be spontaneous at temperatures where metabolism goes on, rather than being caused by use of the system for enzyme production.

Repression and De-repression of Enzyme Synthesis as a Possible Explanation of some Aspects of Drug Action

ACCORDING to recent theories, the induction of enzyme synthesis occurs as a result of the displacement from an enzyme-forming system of a natural repressor1. For example, the synthesis of alkaline phosphatase by E. coli is normally repressed by inorganic phosphate2. De-repression occurs when there is only a small amount of inorganic phosphate in the medium. The repressor theory is in good agreement with most of the observations which have been made on enzyme-inducing systems. The inducer need not be a substrate for the enzyme which is formed, nor need it serve as a template for the formation of new enzymes1.

INTERACTIONS BETWEEN ENZYME-FORMING SYSTEMS DURING ADAPTATION

Experiments on enzymatic adaptations in yeast to galactose and maltose under various conditions are examined. The pertinent facts established may be summarized as follows:- 1. The presence of exogenous nitrogen stimulates the rate of adaptation and raises considerably the attainable level of enzyme activity. 2. This stimulation is absent if the cells are unable to assimilate the added nitrogen. 3. Competitive interactions can be exhibited between two adaptive enzyme systems induced either serially or simultaneously in the same cell. 4. A similar kind of interaction was observed between an adaptive and a so called "constitutive" enzyme. 5. The presence of exogenous nitrogen modifies greatly the nature and extent of the interaction between the enzyme-forming systems. The significance of these results to our understanding of the mechanism of the modification and maintenance of cellular enzymatic constitution is discussed. The validity of the distinction between "constitutive" and "adaptive" enzymes is reexamined in the light of the data presented.