Decay Of Enzyme Activity Research Articles

Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective. The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation. No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction. The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.

On the mechanism of activation of fat cell adenylate cyclase by guanine nucleotides. An explanation for the biphasic inhibitory and stimulatory effects of the nucleotides and the role of hormones.

Adenylate cyclase activity in purified plasma membranes from rat fat cells displays transient kinetic characteristics in the absence and presence of guanyl=5'=yl imidodiphosphate (Gpp(NH)p). Gpp(NH)p causes immediate inhibition of enzyme activity; the inhibitory phase is followed by a slow increase in activity which, depending on incubation temperature, exceeds activity stimulated in the presence of hormones (glucagon, secretin, epinephrine, or adrenocorticotropin). Basal activity displays an initial high rate of activity which decays to a low state of activity within 2 min of incubation. Hormones do not alter the initial rate but prevent the decay in enzyme activity. The inhibitory phase of Gpp(NH)p action and the previously reported (Harwood, J.P., Low, H., and Rodbell, M. (1973) J. Biol. Chem. 248, 6239-6245) inhibitory effects of GTP are abolished by increasing (Mg2+) and pH to 50 mM and 8.5, respectively. Under these conditions, Gpp(NH)p and GTP cause marked stimulation of activity, the stimulatory effect of Gpp(NH)p being greater than that of GTP both in the absence and presence of hormones...

Induction of Phenylalanine Ammonia-lyase in Strawberry Leaf Disks

The increase in phenylalanine ammonia-lyase (PAL) activity in strawberry (Fragaria vesca var. WSU-1232) leaf disks required wounding, sucrose, and light and was cycloheximide-sensitive. In injured leaves and in leaf disks, the highest PAL activity was detected nearest the wounded tissues. Without wounding, no increase in activity was observed when leaves were cultured in sucrose and light.The optimal concentration of sucrose for enzyme activity increase ranged from 0.15 m to 0.4 m. At the suboptimal sucrose concentration, the level of PAL activity was dependent upon the concentration of sucrose. A low but constant level of activity was detected in leaf disks maintained in 0.15 m sucrose and in darkness. Light accelerated the rate of PAL increase but did not change the total level of enzyme activity which was determined by the sucrose concentration.Enzyme activity disappeared rapidly when leaf disks cultured in sucrose and light were transferred to darkness or to water in light. Unlike in Xanthium leaf disks, cycloheximide could not completely inhibit the decay of enzyme activity, suggesting that an inactivating system was synthesized during the induction period, and the activity of the inactivating system increased as the induction period lengthened.The effect of light on accumulation of PAL activity appeared to be linked to photosynthesis. In the presence of 25 mum 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the effect of light on enzyme increase was completely nullified. Addition of 25 mum 3-(3,4-dichlorophenyl)-1,1-dimethylurea to culture medium caused rapid decay of PAL activity from leaf disks which had been previously cultured in sucrose and light. The relation between effect of light and photosynthesis was further demonstrated by the action spectrum. Leaf disks incubated in sucrose and light of different wavelengths exhibited maximum accumulation of PAL activity at two wavelengths (475 nm and 625 nm). Action spectrum for protection against PAL decay exhibited a plateau at 475 to 525 nm and a peak at 625 nm. Action spectra for accumulation and protection against inactivation of PAL activity, therefore, appeared to be very similar to the action spectrum of photosynthesis.

Regulation of Glutamine Synthetase Activity of Hepatoma Tissue Culture Cells by Glutamine and Dexamethasone

Abstract Regulation of glutamine synthetase activity was studied in a glutamine-independent subclone of the hepatoma tissue culture cell line. In cells of this subclone glutamine synthetase activity was elevated by the synthetic corticosteroid hormone, dexamethasone, and depressed by glutamine. The increase in glutamine synthetase activity due to the addition of dexamethasone and that due to removal of glutamine were both inhibited by cycloheximide, indicating that both effects require de novo synthesis of protein. Actinomycin D completely inhibited the increase in glutamine synthetase activity due to addition of dexamethasone but only slightly inhibited the increase in glutamine synthetase activity due to lowering the glutamine concentration. These observations indicate that the action of the hormone requires the synthesis of ribonucleic acid, whereas the main effect of lowering the glutamine concentration is post-transcriptional. When glutamine was added to cells maintained in low glutamine plus dexamethasone, which have maximal glutamine synthetase activity, there was a rapid decay of enzyme activity. The initial rate of decay of glutamine synthetase activity increased with the concentration of glutamine in the medium, reaching a maximum at 5 mm glutamine. At this concentration of glutamine, glutamine synthetase activity decreased to half its initial value in about 2 hours. Actinomycin D slightly inhibited and cycloheximide markedly inhibited the disappearance of glutamine synthetase activity due to glutamine addition. NaF completely inhibited the decay of glutamine synthetase due to glutamine addition. The kinetics of decay of enzyme activity and the effects of inhibitors suggest that glutamine depresses glutamine synthetase activity by accelerating its inactivation or breakdown.

Studies on Rat Liver Phenylalanyl Transfer Ribonucleic Acid Synthetase: II. FURTHER PURIFICATION, SUBSTRATE SPECIFICITY, AND EFFECTS OF SUBSTRATES ON HEAT INACTIVATION

Abstract The phenylalanyl-tRNA synthetase (l-phenylalanine: tRNA ligase [AMP]EC 6.1.1.b) from rat liver has been purified 1500-fold by the addition of hydroxylapatite chromatography to a previously developed procedure. From sodium dodecyl sulfate electrophoresis the enzyme appears to be composed of two types of subunits with molecular weights of 74,500 and 69,000, respectively. Thermal inactivation appears to be associated with breakdown of the larger subunit. The effect of various ligands on the thermal inactivation of the enzyme was investigated. Triphosphates, in general, had a destabilizing effect with adenosine triphosphate (ATP) exerting the greatest influence on the rate of thermal decay of enzyme activity. It seems that the pyrophosphate moiety is responsible for the labilization in these cases since, under the same conditions, sodium pyrophosphate had an even greater effect on the thermal stability of the enzyme than ATP. The presence of magnesium ions reduced the effect of both of these substrates. In the presence of magnesium ion, phenylalanine protected against heat inactivation, but its thermal effect was additive with that of ATP, providing no evidence for the formation of an aminoacyl adenylate. Transfer RNAphe from rat liver and yeast also protected the enzyme against heat inactivation both in the absence and presence of magnesium. Studies with tRNAval, tRNAfmet, tRNAarg, and tRNAphe from Escherichia coli indicated that the amount of protection a tRNA afforded against heat inactivation was reflected in the extent to which it was chargeable by the enzyme, while those tRNAs which were not chargeable but still were bound to the enzyme seemed to strain the enzyme, thus increasing its heat lability.

The Effects of Enzyme Synthesis and Stability and of Deoxyribonucleic Acid Replication on the Cellular Levels of Aspartate Transcarbamylase during the Cell Cycle of Eucaryote Chlorella

Abstract Homogenates of Chlorella pyrenoidosa (strain 7-11-05) contain an organic, low molecular weight (l 1000), heat stable, acid-soluble, Norit A adsorbable compound required for the in vitro stability of aspartate transcarbamylase. Dilution of these homogenates or removal of the stabilizer by either ultrafiltration, gel filtration, ammonium sulfate precipitation, or sucrose density gradient centrifugation resulted in the rapid first order decay of enzyme activity even at 0–3°. The apparent feedback inhibitor, UMP, was the only nucleotide effective as an in vitro stabilizer within physiological concentrations. Although carbamyl phosphate could stabilize the enzyme in vitro, it was eliminated as the possible endogenous stabilizer because carbamyl phosphate is heat labile. The other substrate, aspartate, was ineffective as a stabilizer. In cell cycle studies with synchronous cells of Chlorella, the pattern of aspartate transcarbamylase was observed to parallel the periodic DNA pattern except for a period of gradual enzyme accumulation immediately prior to the step increase in DNA level. Prior to the period of gradual enzyme accumulation, the enzyme level remained constant with a steady state existing between enzyme synthesis and breakdown. The structural gene of the enzyme appeared to be continuously transcribed and the enzyme continuously synthesized so that the enzyme increased from one steady state level to another during the period of gene replication. The gradual increase in activity prior to the step increase in DNA is proposed to be caused by the accumulation of an endogenous stabilizer (e.g. UMP) which reduces the rate of enzyme degradation. This inference is supported by the accumulation of total nucleotide-P and of an endogenous inhibitor of enzyme activity, and by the decreased rate of breakdown of the enzyme in vivo and also in cell homogenates from this period of the cell cycle.

Trehalase activity in dormant and activated spores of Phycomyces blakesleeanus

Heat treatment of Phycomyces sporangiospores, which breaks dormancy, causes a very rapid 10- to 15fold increase in trehalase activity; soon after the heat shock the enzyme activity decays. This phenomenon can be repeated several times by repeating the heat shocks. Prolonging the heat treatment over the minimum required time delays the decay of enzyme activity. Cycloheximide does not prevent the rise in enzyme activity. It is suggested that heat treatment converts temporarily an inactive form of trehalase into an active one. Optimal enzyme activity is obtained at pH 7.5 and the enzyme requires metal ions for maximal activity. The possible role of trehalase in the spore-activation process is discussed.

Acetyl Coenzyme A Carboxylase: III. FURTHER STUDIES ON THE RELATION OF CATALYTIC ACTIVITY TO POLYMERIC STATE

Abstract Catalytic activity of the filamentous polymeric form of avian liver acetyl-CoA carboxylase, (s20,w = 45 to 47 S) undergoes first-order decay in assay reaction mixture (t½ ∼ 10 min at 2°), which is rapidly reversed (l10 s) by addition of tricarboxylic acid activator, i.e. citrate or isocitrate. As previously shown by sucrose density gradient centrifugation, extended exposure to assay reaction mixture leads to the formation of a catalytically inactive 13 to 15 S protomeric species of the enzyme. If avidin, the biotin-binding protein from egg white, is added following this decay, reactivation by citrate is blocked completely. However, when citrate is present in the assay reaction mixture, thereby maintaining the enzyme in the polymeric state, inactivation by avidin is prevented. The presence of avidin in the assay reaction mixture does not alter the t½ for activity decay, although reactivation is no longer possible upon addition of citrate. Only that fraction of carboxylase activity remaining can be protected by citrate from further erosion of activity and inactivation by avidin. Thus, the characteristics and kinetics of the decay of catalytic activity are compatible with a transition from an avidin-insensitive, active polymeric species to an avidin-sensitive, catalytically inactive form. Consistent with a polymer (intrinsic viscosity, 83) to protomer (intrinsic viscosity, 11.5) transition, the decay of enzyme activity is tightly coupled to an apparent first order (t½ = 10 min) decline in viscosity, t½ (activity) = t½ (viscosity). Citrate addition results in the rapid restoration of carboxylase activity and an abrupt rise in viscosity, equivalent to the percentage of reactivation. Tight coupling of reversible loss of enzyme activity to depolymerization is further supported by the observation that Mg2+ ATP and HCO3-, which carboxylate the enzyme, promote a rapid loss of catalytic activity (t½ = ½ min), the formation of the avidin-sensitive species, as well as, a rapid decrease in viscosity. The presence of carboxyl acceptor (acetyl-CoA), which retards activity decay (t½ = 7½ min), and susceptibility to avidin, also delays the fall in viscosity. It is proposed that the loss of catalytic activity and formation of the avidin-sensitive species reflects a polymer to protomer transition.

Factors affecting acid phosphatase activity in exponential and synchronized L5178Y mouse leukemia cells

The effects of the following factors on acid phosphatase activity in L5178Y cells were studied: inhibitors of RNA, DNA, and protein synthesis, phosphate concentration, growth cycle and cell cycle. Puromycin, high and low phosphate concentrations, and high cell density caused a decrease in enzyme activity while inhibition of DNA synthesis and RNA synthesis caused an increase of activity. During log phase, the cells displayed a constant level of activity which declined to a lower level as the cells entered the stationary phase. In synchronized cells the enzyme activity varied periodically: a minimum in G1 with peaks in late G1-early S and again in late S-G2. Treatment of cells in the log phase with puromycin caused a linear decay of enzyme activity. In synchronized cells, puromycin also caused a linear decay of enzyme activity at all points in the cell cycle and enzyme activity decayed to levels well below those of controls. From this, it is concluded that the cell regulates its level of acid phosphatase activity by varying the rate of synthesis while the rate of decay remains constant throughout the cell cycle.

Turnover Rate of Hepatic 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase as Determined by Use of Cycloheximide

Abstract Both rat liver cholesterol synthesis and the primary controlling enzyme in this synthesis, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) are known to exhibit a circadian rhythm of activity. Rats fed ad libitum and housed under normal conditions (i.e. lights on at 6 a.m. and off at 6 p.m.) showed a maximum activity of HMG-CoA reductase at midnight and a minimum activity at noon. Changing the lighting conditions such that there was still a 12-hour light-dark cycle per day affected the timing but not the amplitude of the rhythm. The maximum reductase activity always occurred 6 hours after the onset of the dark period. Cycloheximide (1.3 µg per g body weight) caused an exponential decay of liver microsomal HMG-CoA reductase activity for a 9-hour period following the injection. On a normal lighting schedule this decay rate was the same when measured from 9 a.m., 9 p.m., or 2 a.m. The half-life (t½) of the enzyme was calculated to be approximately 4.2 hours. The rate of decay of enzyme activity measured from 2 a.m. was the same for untreated control rats fed ad libitum and for rats treated with cycloheximide. Consequently, the circadian rhythm can be explained as due solely to changes in the rate of synthesis of HMG-CoA reductase.