Retrograde movement of membrane lipids from the Golgi apparatus to the endoplasmic reticulum of perforated cells: evidence for lipid recycling.
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Animals↗
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Results for “Substrate Cycling”
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Kinetic characterization of rat tissue kallikrein using N alpha-substituted arginine 4-nitroanilides and N alpha-benzoyl-L-arginine ethyl ester as substrates.
Hydrolysis of seven N alpha-substituted L-arginine 4-nitroanilides: benzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 microM for p-nitroanilides and 50 to 190 microM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 microM for p-nitroanilides and 200 to 1800 microM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 +/- 2 microM; Vmax = 10.42 +/- 0.28 microM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 +/- 2 microM; Vmax = 3.21 +/- 0.11 microM/min), while Tos-Arg-Nan (Km = 29 +/- 2 microM; Vmax = 0.10 +/- 0.002 microM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 +/- 15 microM; Vmax = 121.3 +/- 7.6 microM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.
Animals↗
Recycling of alpha-D-ribose 1-phosphate for nucleoside interconversion.
Mobilization of the ribose moiety and of the amino group of guanosine may be realized in rat liver extract by the concerted action of purine nucleoside phosphorylase and guanase. Ribose 1-phosphate formed from guanosine through the action of purine nucleoside phosphorylase acts as ribose donor in the synthesis of xanthosine catalyzed by the same enzyme. The presence of guanase, which irreversibly converts guanine to xanthine, affects the overall process of guanosine transformation. As a result of this purine pathway, guanosine is converted into xanthosine, thus overcoming the lack of guanosine deaminase in mammals. Furthermore, in rat liver extract the activated ribose moiety stemming from the catabolism of purine nucleosides can be transferred to uracil and, in the presence of ATP, used for the synthesis of pyrimidine nucleotides; therefore, purine nucleosides can act as ribose donors for the salvage of pyrimidine bases.
Animals↗
Regulation of energy balance and adiposity: a model with new approaches.
Obesity etiology and treatment have been fraught with disappointment for researchers, because the mechanisms regulating fuel homeostasis and adiposity are incompletely understood. It can now be hypothesized in the light of new evidences that the control of body weight and composition depends upon an axis with three interrelated and self-controlled components: 1) food intake; 2) nutrient turnover and thermogenesis and 3) body fat stores, all of which underly complex feedback mechanisms. This approach considers two of the most relevant recent findings in the field (leptin and beta 3-adrenoceptors), adding new views to previous metabolic models of obesity. This perspective supplies some additional clues to the understanding of body composition regulation as well as the potential involvement of genetic and hypothalamic disorders in the onset of obesity.
Adipose Tissue↗
Production of cyclomaltodextrin glucanotransferase from an alkalophilic Bacillus sp. in rice and corn flakes.
Cyclomaltodextrin glucanotranferase (CGTase) catalyzes the degradation of starch to form alpha-, beta- and gamma-cyclodextrin. Based on cyclodextrin formation, an alkalophilic Bacillus sp. ATCC 21783 was used for its high CGTase production ability, using corn and rice flakes as substrates. Maximum enzyme production was achieved after 96 hours at pH 9.0, temperature 37 degrees C, and 1% (w/v) of either substrate together with the addition of trub. The specific enzyme activity was determined by High Pressure Liquid Chromatography (HPLC) and expressed as using International Units based on total cyclodextrin formation. Optimum conditions for this determination were studied, finding that the best results are obtained at pH 5.0, 7.0 and 9.0, temperature 55 degrees C and 3 hours of incubation in 1% (w/v) of rice flakes as starch source.
Bacillus↗
[Substrate metabolism in untreated and treated thyrotoxicosis].
Accelerated metabolism is a hallmark of thyrotoxicosis, but the underlying biochemical mechanisms are incompletely understood. In order to elucidate these metabolic events further, we studied 12 patients with newly diagnosed diffuse (10 patients) or nodular (two patients) toxic goitre (ten women, two men; age 42.8 +/- 3.2 yr; BMI: 21.6 +/- 0.7 kg/m2) before ("TOX") and after ("TRE") 11.2 +/- 1.0 weeks treatment with methimazole and compared these patients to a control group ("CTR") of 11 subjects (nine women, two men; age 40.5 +/- 3.9 yr; BMI 22.5 +/- 1.0 kg/m2). All were studied for three hours in the basal state, using indirect calorimetry, isotope dilution for measurement of glucose turnover and the forearm technique for assessment of muscle metabolism. Prior to treatment patients with thyrotoxicosis were characterized by: Increased (p < 0.05) levels of T3 (3.75 +/- 0.23 [TOX], 1.89 +/- 0.08 [TRE] and 1.75 +/- 0.11 [CTR] nmol/l), resting energy expenditure (130.5 +/- 3.5 [TOX], 107.7 +/- 2.7 [TRE] and 106.3 +/- 3.1 [CTR] percent of predicted), protein oxidation (0.67 +/- 0.03 [TOX], 0.54 +/- 0.06 [TRE] and 0.46 +/- 0.05 [CTR] mg/kg/min), lipid oxidation (1.34 +/- 0.08 [TOX], 1.00 +/- 0.06 [TRE] and 1.02 +/- 0.04 [CTR] mg/kg/min), endogenous glucose production (2.51 +/- 0.13 [TOX], 1.86 +/- 0.12 [TRE] and 1.85 +/- 0.12 [CTR] mg/kg/min), non-oxidative glucose turnover (1.28 +/- 0.16 [TOX], 0.75 +/- 0.18 [TRE] and 0.71 +/- 0.11 [CTR] mg/kg/min) and a 50% increase in total forearm blood flow. Glucose oxidation (1.23 +/- 0.09 [TOX], 1.13 +/- 0.10 [TRE] and 1.13 +/- 0.09 [CTR] mg/kg/min), exchange of substrates in the muscles of the forearm and circulating levels of insulin, C-peptide, growth hormone or glucagon were not influenced by hyperthyroidism. Propranolol (20 mg thrice daily) given to seven of the patients for two days did not affect circulating levels of thyroid hormones, energy expenditure or glucose turnover rates. These results suggest that all major fuel sources contribute to the hypermetabolism of thyrotoxicosis and that augmented non-oxidative glucose metabolism may further aggravate the condition. All abnormalities recede with medical treatment of the disease.
Adult↗
Hsp70 chaperone systems: diversity of cellular functions and mechanism of action.
Hsp70 chaperone systems play an essential role in the life cycle of many proteins not only in an hostile environment but also under normal growth conditions. In the course of evolution the diversification of functions was accompanied by an amplification of components of the Hsp70 system. Here strategies are reviewed how different Hsp70 systems work independently or cooperate with each other in a functional network to perform their housekeeping tasks even under stress conditions. We further discuss how co-chaperones which act as targeting factors regulate the cycle of substrate binding and release upon which the Hsp70 chaperone activity depends.
HSP70 Heat-Shock Proteins↗
Subtleties in control by metabolic channelling and enzyme organization.
Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.
Energy Metabolism↗
Removal of organic carbon, nitrogen and phosphorus in sequential batch reactors integrating the aerobic/anaerobic processes.
The performance of a bench-scale apparatus composed of two sequential batch reactors (SBR), forming an anaerobic/aerobic treatment system, was evaluated as to its potential use for biological removal of organic matter and nutrients. Both the reactors with 12.5 L of useful volume each were operated for the batch cycles of 12 hours receiving 8.0 L of substrate in each cycle. The first reactor (SBRAn) fed with synthetic substrate simulating domestic sewage was meant to remove the largest fraction of carbonic matter and to promote the substrate ammonification. The second reactor (SBRAe) was operated under alternating aerobic and anoxic conditions to establish conditions for achieving nitrification; denitrification and biological phosphate removal in the same batch cycle. Sodium acetate was used as an external carbon source for phosphate removal. Processes monitoring included the analyses of: COD, NTK-N, NH4(+)-N, NO2(-)-N, NO3(-)-N, PO4(3-)-P, alkalinity, volatile acids, pH, redox potential total, fixed and volatile solids. Under the operating conditions imposed, the system exhibited high performance in removing organic matter (COD), nitrogen and phosphorus, producing effluent with COD, NTK-N and PO4(3-)-P concentrations lower than 50.0 mg/L, 4.0 mg/L and 1.0 mg/L, respectively.
Bacteria, Aerobic↗
[Substrate inhibition in the tricarboxylic acid cycle].
It has been shown in the experiments on rat liver mitochondria under glucose hexo-kinase load that excess of substrates of (1-20 mM) pyruvate, acetate, propionate, pent-4-enoate and malate may induce oxidation of NAD(P)H and inhibition of mitochondrial respiration (by 20-50% and more) due to a decreased rate of hydrogen production by tricarboxylic acid cycle. It has been concluded from the analysis of mathematical models and metabolite-testings which remove this inhibition that for pyruvate and acetate this inhibition is an autocatalytic one. It is related to a decreased level of CoA and oxaloacetate due to the formation of "traps" such as acetyl-CoA and alpha-kotoglutarate. For propionate and pent-4-enoate in the bicarbonate-free medium suppression of the flux in the cycle is concerned with a decreased level of CoA, acetyl-CoA and succionoyl CoA due to the accumulation of propionyl-CoA. It seems to be also concerned with the inhibition of citrate-synthetase and alpha-ketoglutarate-dehydrogenase by propionyl-CoA. Malate (in the presence of malonate) can inhibit respiration at the expense of direct inhibition of citrate-synthetase.
Acetates↗
[Hysteresis, alternative stationary states and auto-oscillations in an open futile cycle one of the reactions of which is substrate inhibited].
In connection with the problem of regulation of futile (energy-dissipating) cycles in cell metabolism, a kinetic model has been investigated of an open cycle S1 (see article) S2, in which one of the enzymes (E-) is inhibited by the excess of its substrate S2. The quasi-stationary net velocity of the utilization of substrate S1 in the cycle as a function of its concentration is shown to be of a hysteretic character. Owing to this the alternative stationary states and self-oscillations may occur in the cycle. Under certain conditions the transition from one alternative state to another may reverse the direction of the net flux of conversion from S1 to S2 or vice versa. The self-oscillations are associated with a periodical change in the net flux direction. It is suggested the participation of glycogen (starch) in the self-oscillatory mechanism of the futile cycle formed by the phosphofructokinase and fructose bisphosphatase reactions may give rise to oscillations with the period of 10(3)-10(4) min, which may serve as the basis for the cell clock.
Adenylate Kinase↗
Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities.
Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40(MO15) have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40(MO15) only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21(CIP1), p27(KIP1), p57(KIP2), p16(INK4a), and p18(INK4c), could block phosphorylation by p40(MO15) but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40(MO15) activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.
CDC2-CDC28 Kinases↗
The derivation of second degree rate equations arising from two-substrate, two-product enzyme catalysed reactions whose catalytic cycle is branched.
A selection of two-substrate, two-product enzyme catalysed reactions with alternate catalytic cycles and second degree rate equations was examined by the computer program described by Kinderlerer and Ainsworth (1976). It is shown that the numerator terms of the rate equations are particularly simple, easy to derive and capable of creating a broad division of the mechanisms considered. THE NUMERATOR TERMS ALSO HAVE THE ADVANTAGE OF BEING UNAFFECTED BY DEAD END INHIBITION OR THE PRESENCE OF Theorell-Chance reactions. It is then shown that the relatively small group of mechanisms, isolated by the numerator analysis, can be distinguished from each other by considering the denominator terms of the rate equation that are second degree in the concentration of the varied substrate.
Catalysis↗
Upper processing stages of the perception-action cycle.
The neural substrate for behavioral, cognitive and linguistic actions is hierarchically organized in the cortex of the frontal lobe. In their methodologically impeccable study, Koechlin et al. reveal the neural dynamics of the frontal hierarchy in behavioral action. Progressively higher areas control the performance of actions requiring the integration of progressively more complex and temporally dispersed information. The study substantiates the crucial role of the prefrontal cortex in the temporal organization of behavior.
Animals↗
Triiodothyronine administration affects urea synthesis in rats.
The purpose of the present study was to elucidate the mechanism by which thyroid hormone alters urea synthesis. The relative importance of urea cycle enzyme activities, substrate levels or the levels of urea cycle intermediates on urea production was investigated in a set of four experiments in which rats were fed a diet supplemented with 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) and treated with triiodothyronine (T3). Compared with that of normal or control rats, the plasma level of T3 was significantly lower in rats fed a diet containing PTU only and elevated in rats given PTU + T3. Urinary excretion of urea and the liver content of ornithine in rats given PTU + T3 were significantly lower than in rats given PTU only. The liver level of ornithine was closely correlated to the excretion of urea in the present study. Fractional rates of protein synthesis in liver, kidney and small intestine were lower in the hypothyroid group. However, most free amino acid concentrations, except ornithine in liver and plasma and the activity of hepatic argininosuccinate synthetase (EC 6.3.4.5) of hypothyroid rats, were significantly reduced as compared with those of control or hyperthyroid rats. The results indicate that the increased hepatic ornithine content in the hypothyroid rats may be one of the regulatory factors causing changes in urea synthesis.
Administration, Oral↗
Ectopic expression of cyclin D1 but not cyclin E induces anchorage-independent cell cycle progression.
Normal fibroblasts are dependent on adhesion to a substrate for cell cycle progression. Adhesion-deprived Rat1 cells arrest in the G1 phase of the cell cycle, with low cyclin E-dependent kinase activity, low levels of cyclin D1 protein, and high levels of the cyclin-dependent kinase inhibitor p27kip1. To understand the signal transduction pathway underlying adhesion-dependent growth, it is important to know whether prevention of any one of these down-regulation events under conditions of adhesion deprivation is sufficient to prevent the G1 arrest. To that end, sublines of Rat1 fibroblasts capable of expressing cyclin E, cyclin D1, or both in an inducible manner were used. Ectopic expression of cyclin D1 was sufficient to allow cells to enter S phase in an adhesion-independent manner. In contrast, cells expressing exogenous cyclin E at a level high enough to overcome the p27kip1-imposed inhibition of cyclin E-dependent kinase activity still arrested in G1 when deprived of adhesion. Moreover, expression of both cyclins D1 and E in the same cells did not confer any additional growth advantage upon adhesion deprivation compared to the expression of cyclin D1 alone. Exogenously expressed cyclin D1 was down-regulated under conditions of adhesion deprivation, despite the fact that it was expressed from a heterologous promoter. The ability of cyclin D1-induced cells to enter S phase in an adhesion-independent manner disappears as soon as cyclin D1 proteins disappear. These results suggest that adhesion-dependent cell cycle progression is mediated through cyclin D1, at least in Rat1 fibroblasts.
Animals↗
Fatty acid oxidation, substrate shuttles, and activity of the citric acid cycle in hepatocellular carcinomas of varying differentiation.
Fatty acid oxidation, reconstituted substrate shuttles, and the activity of the citric acid cycle were studied in mitochondria isolated from Becker transplantable hepatocellular carcinoma H-252 AND Host livers, and the results were compared with those obtained with Morris hepatomas 7288CTC and 5123C. Whereas the activities of the malate-aspartate and the alpha-glycerophosphate shuttles were only slightly lower than those of host livers, the activity of the fatty acid shuttle was much lower in H-252 mitochondria. Oxygen uptake and CO2 production associated with the oxidation of fatty acids was much lower in tumors H-252 and 7288CTC, compared with host livers, whereas tumor 5123C mitochondria show a high capacity to oxidize fatty acids. Ketogenesis and beta-hydroxybutyrate dehydrogenase activity were also lower in tumor H-252 mitochondria. However, neither oxygen uptake associated with the oxidation of other respiratory substrates nor CO2 production from succinate or malate was strikingly elevated in these tumors. These factors suggest that the respiratory phosphorylation chain and activity of the citric acid cycle are fully functional in tumors H-252 and 7288CTC. The defects responsbile for the lower rates of fatty acid oxidation in these tumors probably involves the beta-oxidation pathway, as well as the activation of fatty acids. The impairment of fatty acid oxidation may explain the lower activity of the reconstituted fatty acid shuttle for transporting reducing equivalents into H-252 mitochondria. The different properties with regard to fatty acid oxidation in Morris hepatoma 5123C, compared with those in Becker H-252- AND Morris hepatoma 7288CTC, may reflect the different extent of differentiation in these tumors, the former being a slow-growing, well-differentiated tumor, whereas the latter represent tumors that are less differentiated and of more rapid growth rate.
Animals↗
Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids.
The monoclonal antibody solution hybridization assay is a novel enzyme immunoassay for detection of RNA with a biotinylated DNA probe. To increase the sensitivity of this test, a fluorescent substrate and an enzymatic amplification cycling system were compared with a conventional colorigenic substrate for alkaline phosphatase. The fluorescent, cycling, and colorigenic substrates detected, respectively, 10, 10, and 100 amol of unbound alkaline phosphatase in 2 h. With a prolonged incubation period of 16.6 h, the conventional substrate measured 10 amol of the enzyme. In the immunoassay for RNA detection, the fluorescence and cycling assays were faster than that using the colorigenic substrate and reached an endpoint sensitivity of 3.2 pg/ml (0.16 pg per assay) of cRNA. However, longer incubation periods (16.6 h) for optimal generation of the colorigenic product led to a comparable level of sensitivity for the conventional substrate.
Biotin↗