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1.
Substitution of A1-glycine of insulin by L-amino acids yields in analogues with low biological activity. With D-amino acids in A1 biological activity is essentially retained. Synthesis of [A1-L-tryptophan]- and [A1-D-tryptophan]-insulin should provide information about the position of the side chains of L- and D-amino acids relative to A19-tyrosine, e.g. by evaluation of intramolecular resonance energy transfer between the fluorescent side chains. [A1-D-Tryptophan]-insulin exhibits full biological activity.  相似文献   

2.
The results are presented from receptor binding studies and isolated fat cell assays on A1-(2-nitro-4-trimethylammoniophenyl)insulin. In the former tests, the derivative is shown to have properties similar to native insulin and the fat cell assays afford a value of 76 +/- 4%. The importance of a charged, hydrophilic moiety attached at A1-glycine is discussed in the light of the above findings.  相似文献   

3.
Mitochondrial extracts of dog, cat, rat and mouse liver contain two forms of alanine-glyoxylate aminotransferase (EC 2.6.1.44): one, designated isoenzyme 1, has mol.wt. approx. 80 000 and predominates in dog and cat liver; the other, designated isoenzyme 2, has mol.wt. approx. 175 000 and predominates in rat and mouse liver. In rat and mouse liver, isoenzyme 1 activity was increased by the injection in vivo of glucagon, but not isoenzyme 2 activity. Isoenzyme 1 was purified and characterized from liver mitochondrial extracts of the four species. Both rat and mouse enzyme preparations catalysed transamination between a number of L-amino acids and glyoxylate, and with L-alanine as amino donor the effective amino acceptors were glyoxylate, phenylpyruvate and hydroxypyruvate. In contrast, both dog and cat enzyme preparations were specific for L-alanine and L-serine with glyoxylate, and used glyoxylate and hydroxypyruvate as effective amino acceptors with L-alanine. Evidence that isoenzyme 1 is identical with serine-pyruvate aminotransferase (EC 2.6.1.51) was obtained. Isoenzyme 2 was partially purified from mitochondrial extracts of rat and mouse liver. Both enzyme preparations were specific for L-alanine and glyoxylate. On the basis of physical properties and substrate specificity, it was concluded that isoenzyme 2 is a separate enzyme. Some other properties of isoenzymes 1 and 2 are described.  相似文献   

4.
The acute effects of physiological levels of leptin (10 ng ml(-1)) and insulin (20 microU ml(-1)) on hepatic gluconeogenesis and ketogenesis were compared. Leptin or insulin alone decreased (p<0.05) the activation of hepatic glucose, L-lactate and urea production from L-alanine. However, the hepatic glucose production was not modified if leptin was combined with insulin. These results indicated that both, i.e. leptin and insulin, could promote a non-additive reduction in the rate of catabolism of L-alanine. However, in contrast with insulin (p<0.05), leptin did not inhibit the activation of hepatic glucose production from pyruvate or glycerol. On the other hand, activation of hepatic production of acetoacetate and beta-hydroxybutyrate from octanoate was not affected by leptin or insulin. Thus, our data demonstrate that the acute effect of leptin on hepatic metabolism was partially similar to insulin (activation of glucose production from L-alanine and activation of acetoacetate or beta-hydroxybutyrate production from octanoate) and partially different from insulin (activation of glucose production from pyruvate or glycerol).  相似文献   

5.
We integrated biological experimental data with mathematical modelling to gain insights into the role played by L-alanine in amino acid-stimulated insulin secretion (AASIS) and in D-glucose-stimulated insulin secretion (GSIS), details important to the understanding of complex β-cell metabolic coupling relationships. We present an ordinary differential equations (ODEs) based simplified kinetic model of core metabolic processes leading to ATP production (glycolysis, TCA cycle, L-alanine-specific reactions, respiratory chain, ATPase and proton leak) and Ca2+ handling (essential channels and pumps in the plasma membrane) in pancreatic β-cells and relate these to insulin secretion. Experimental work was performed using a clonal rat insulin-secreting cell line (BRIN-BD11) to measure the consumption or production of a range of important biochemical parameters (D-glucose, L-alanine, ATP, insulin secretion) and Ca2+ levels. These measurements were then used to validate the theoretical model and fine-tune the parameters. Mathematical modelling was used to predict L-lactate and L-glutamate concentrations following D-glucose and/or L-alanine challenge and Ca2+ levels upon stimulation with a non metabolizable L-alanine analogue. Experimental data and mathematical model simulations combined suggest that L-alanine produces a potent insulinotropic effect via both a stimulatory impact on β-cell metabolism and as a direct result of the membrane depolarization due to Ca2+ influx triggered by L-alanine/Na+ co-transport. Our simulations indicate that both high intracellular ATP and Ca2+ concentrations are required in order to develop full insulin secretory responses. The model confirmed that K+ ATP channel independent mechanisms of stimulation of intracellular Ca2+ levels, via generation of mitochondrial coupling messengers, are essential for promotion of the full and sustained insulin secretion response in β-cells.  相似文献   

6.
Des-Gly-A-chain-tetra-S-sulphonate was prepared by Edman degradation following two different routes. A) Via complete reaction of A-chain from bovine insulin with 150 equivalents of phenylisothiocyanate in pyridine/water and trifluoroacetic acid cleavage of the resulting phenylthiocarbamoyl A-chain. B) Via reaction of bovine insulin with about 20 equivalents of phenylisothiocyanate until a substitution degree of 2.3-2.5 was reached, trifluoroacetic acid cleavage of the crude derivatives and oxidative sulphitolysis of the resulting desaminoacyl insulins. Preparative electrophoresis (pH 2) or ion exchange chromatography using DEAE-Sephadex gave des-Gly-A-chain in a yield of 60-65% of theory according to method B, containing less than 1% of glycine. Des-GlyA1-insulin was prepared by combination with 0.67 equivalents of B-chain-bis-S-sulphonate and isolated in yields of 5-13%, based on B-chain, after gel filtration (pH 8) and ion exchange chromatography (CM-cellulose, pH 3-2). The electrophoretically (pH 2 and 8.6) homogeneous analogue did not crystallize in the presence of zinc ions. Its blood sugar lowering potency is 10-25%, its in vitro insulin activity (fat cell assay) only 1-2%. The immunoreactivity against anti-insulin sera in different test systems is markedly reduced. There are clear differences between the CD-spectra of des-Gly-insulin and insulin, indicating a loss of ordered secondary structure. From the results it is concluded that structure-stabilizing non covalent bonds are abolished by the removal of the invariant A1-glycine. This leads to conformational alterations which cause the far-going inactivation of the molecule.  相似文献   

7.
Des-(B25-B30)-hexapeptide-insulin with B23-glycine replaced by D-alanine was prepared by a combination of enzymic and non-enzymic syntheses. The purified product was homogeneous in polyacrylamide-gel electrophoresis and could be crystallized. The biological activity in vivo of crystalline [B23-D-Ala]des-(B25-B30)-hexapeptide-insulin was determined as 58% of that of standard pig insulin (27 i.u./mg).  相似文献   

8.
1. Lactation is associated with an increase in the arterial blood concentration of L-alanine and L-glutamate, but a decrease in that of L-glutamine compared with the corresponding values for virgin rats. 2. Virgin rats fed a 'cafeteria diet' that induces hyperphagia have increased arterial concentrations of L-alanine, L-glutamate and L-glutamine. During lactation L-alanine and L-glutamate concentrations are even higher. 3. The removal of L-alanine is decreased in hepatocytes from lactating rats fed either a chow or cafeteria diet. 4. Measurements of arteriovenous differences across lactating mammary glands indicate that appreciable amounts of L-glutamine and L-alanine are extracted by the gland. 5. A high proportion of the L-alanine metabolized by isolated acini from fed lactating rats is converted into lipid. 6. Metabolism of L-alanine in acini from starved lactating rats is limited by the activity of pyruvate dehydrogenase. 7. It is concluded that L-alanine and certain other amino acids taken up by the gland in excess of the requirements for protein synthesis can be converted into lipid.  相似文献   

9.
Adenosine is known to influence different kinds of cells, including beta-cells of the pancreas. However, the role of this nucleoside in the regulation of insulin secretion is not fully elucidated. In the present study, the effects of adenosine A(1) receptor antagonism on insulin secretion from isolated rat pancreatic islets were tested using DPCPX, a selective adenosine A(1) receptor antagonist. It was demonstrated that pancreatic islets stimulated with 6.7 and 16.7 mM glucose and exposed to DPCPX released significantly more insulin compared with islets incubated with glucose alone. The insulin-secretory response to glucose and low forskolin appeared to be substantially potentiated by DPCPX, but DPCPX was ineffective in the presence of glucose and high forskolin. Moreover, DPCPX failed to change insulin secretion stimulated by the combination of glucose and dibutyryl-cAMP, a non-hydrolysable cAMP analogue. Studies on pancreatic islets also revealed that the potentiating effect of DPCPX on glucose-induced insulin secretion was attenuated by H-89, a selective inhibitor of protein kinase A. It was also demonstrated that formazan formation, reflecting metabolic activity of cells, was enhanced in islets exposed to DPCPX. Moreover, DPCPX was found to increase islet cAMP content, whereas ATP was not significantly changed. These results indicate that adenosine A(1) receptor blockade in rat pancreatic islets potentiates insulin secretion induced by both physiological and supraphysiological glucose concentrations. This effect is proposed to be due to increased metabolic activity of cells and increased cAMP content.  相似文献   

10.
Temporal inhibition of protein synthesis with cycloheximide prevents subsequent insulin, but not serum-stimulated DNA synthesis in G1-arrested chick embryo fibroblasts (CEF). The inhibition is measured by the incorporation of 3H-thymidine into acid insoluble material and confirmed by chemical estimate of the DNA content of inhibited and uninhibited cells. Cycloheximide treatment is without effect if the cell cultures are maintained at 4 degrees C while exposed to the drug. Several alpha-keto acids (pyruvate, oxaloacetate, alpha-ketobutyrate) at 0.5-1 mM concentrations restore DNA synthesis in previously inhibited cells when combined with insulin. L-alanine (D-alanine is inert) is even more effective than the keto acids in stimulating DNA synthesis after cycloheximide treatment. Glucose transport was unaffected by cycloheximide treatment while lactate levels in medium from inhibited, insulin-stimulated CEF were reduced 70% compared to uninhibited counterparts. We speculate that cycloheximide treatment may lead to the decay of a glycolytic enzyme which compromises the ability of inhibited cells to synthesize pyruvate from glucose, and thus induces an exogenous requirement for alpha-keto acid or L-alanine. A serum component(s) with a molecular weight of about 100 permitted insulin-stimulated DNA synthesis in inhibited cells.  相似文献   

11.
The role of substrate availability in the regulation of gluconeogenesis in isolated rat hepatocytes was studied using [U-14C]alanine as a tracer in the presence of different concentrations of L-alanine in the incubation medium. At low alanine concentrations (0.5 mM) insulin decreased the 14C incorporation into the glucose pool and increased the incorporation of tracer carbons into the protein and lipid pools and into CO2. The net radioactivity lost from the glucose pool was only a small percentage of the total increase in the activity of the protein, lipid, CO2, or glycogen pools, supporting the notion that the effect of insulin in diminishing gluconeogenesis is secondary to its effects on pathways using pyruvate. At higher concentrations of alanine (2.5, 5.0, and 10.0 mM) in the incubation medium insulin increased the movement of alanine carbons into protein and glucose. This suggests that at higher substrate concentrations the ability of the liver to synthesize proteins is overwhelmed and the pyruvate carbons are forced into the gluconeogenesis pathway. These results were further confirmed by using [U-14C]lactate. The increases in observed specific activity of glucose following insulin administration would not be possible if insulin acted by affecting the activity of any enzyme directly involved in the formation or utilization of pyruvate, most of which have been proposed as sites of insulin action. Data presented show that insulin "inhibits" gluconeogenesis by affecting a change in substrate availability.  相似文献   

12.
L-alanine:4,5-dioxovalerate transaminase activity and chlorophyll levels were estimated in lead and mercury treated Bajra seedlings. The enzyme activity increased with age upto 2nd day of germination and decreased on consequent days, where as the chlorophyll content increased with age upto 4th day and remained constant on day 5. Both the metals have no effect on L-alanine: 4,5-dioxovalerate transaminase activity but reduced chlorophyll levels. In vitro incubation of the enzyme with metal solutions showed that the enzyme activity was inhibited by mercury, while lead had no effect. Studies on sub-cellular localization of the L-alanine:4,5-dioxovalerate transaminase showed that it is present in all fractions. The non-correlation between L-alanine: 4,5-dioxovalerate transaminase activity and chlorophyll synthesis is evident from different activity profiles with age and response to heavy metal treatment in the seedlings. Hence, our results suggest the non-involvement of L-alanine:4,5-dioxovalerate transaminase in chlorophyll synthesis in bajra seedlings.  相似文献   

13.
Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.  相似文献   

14.
Insulin regulation of amino acid transport across the cell membrane was studied in a variety of mesenchymal cell directly isolated from avian and mammalian tissues or collected from confluent cultures. Transport activity of the principal systems of mediation in the presence and absence of insulin was evaluated by measuring the uptake of representative amino acids under conditions approaching initial entry rates. Insulin enhanced the transport rate of substrate amino acids from the A system(alpha-aminoisobutyric acid, L-proline, glycine, L-alanine and L-serine) in fibroblasts and osteoblasts from chick-embryo tissues, in mesenchymal cells (fibroblasts and smooth muscle cells) from immature rat uterus, in thymic lymphocytes from young rats and in chick-embryo fibroblasts from confluent secondary cultures. In these tissues, the uptake of amino acid substrates of transport systems L and Ly+ (L-leucine, L-phenylalanine, L-lysine) was not affected by the presence of the hormone. No insulin control of amino acid transport was detected in chick-embryo chondroblasts and rat peritoneal macrophages. These observations identify the occurrence of hormonal regulatory patterns of amino acid transport for different mesenchymal cells types and indicate that these properties emerge early during cell differentiation.  相似文献   

15.
Amino acid transport in horse erythrocytes is regulated by three co-dominant allelomorphic genes coding for high-affinity transport activity (system asc1), low-affinity transport activity (system asc2) and transport-deficiency, respectively. The asc systems are selective for neutral amino acids of intermediate size, but unlike conventional system ASC, do not require Na+ for activity. In the present series of experiments we have used a combined kinetic and genetic approach to establish that dibasic amino acids are also asc substrates, systems asc1 and asc2 representing the only mediated routes of cationic amino acid transport in horse erythrocytes. Both transporters were found to exhibit a strong preference for dibasic amino acids compared with neutral amino acids of similar size. Apparent Km values (mM) for influx via system asc1 were L-lysine (9), L-ornithine (27), L-arginine (27), L-alanine (0.35). Corresponding Vmax estimates (mmol/l cells per h, 37 degrees C) were L-lysine (1.65), L-ornithine (2.15), L-arginine (0.54), L-alanine (1.69). Apparent Km values for L-lysine and L-ornithine influx via system asc2 were approximately 90 and greater than 100 mM, respectively, with Vmax values greater than 2 and greater than 1 mmol/l cells per h, respectively. Apparent Km and Vmax values for L-alanine uptake by system asc2 were 14 mM and 6.90 mmol/l cells per h. In contrast, L-arginine was transported by system asc2 with the same apparent Km as L-alanine (14 mM), but with a 77-fold lower Vmax. This dibasic amino acid was shown to cause cis- and trans-inhibition of system asc2 in a manner analogous to its interaction with system ASC, where the side-chain guanidinium group is considered to occupy the Na+-binding site on the transporter. Concentrations of extracellular L-arginine causing 50% inhibition of zero-trans L-alanine influx and half-maximum inhibition of L-alanine zero-trans efflux were 14 mM (extracellular L-alanine concentration 15 mM) and 3 mM (intracellular L-alanine concentration 15.5 mM), respectively. We interpret these observations as evidence of structural homology between the horse erythrocyte asc transporters and system ASC. Physiologically, intracellular L-arginine may function as an endogenous inhibitor of system asc2 activity.  相似文献   

16.
In the present study, we examined the possibility that the excess heme generation within mitochondria may provide a local concentration, sufficient to inhibit the activity of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid biosynthesis in mammalian system. This was accomplished by assaying together L-alanine:4,5-dioxovalerate transaminase and heme synthetase activities in intact mitochondria isolated from rat liver. Endogenous heme in intact mitochondria has been generated in excess, by increasing the concentration of the substrate of heme synthetase. Our studies showed that the activity of L-alanine:4,5-dioxovalerate transaminase decreased as the rate of heme formation increased. In intact mitochondria, almost 50% inhibition of alanine:4,5-dioxovalerate transaminase was obtained with 4.0 mumole of heme generation. We conclude that end product inhibition of L-alanine:4,5-dioxovalerate transaminase by hemin, which was proposed in earlier report by us (FEBS Letter (1985), 189, 129), is an important physiological mechanism for the regulation of hepatic heme biosynthesis.  相似文献   

17.
An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [3H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and α-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine and D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.  相似文献   

18.
Available information on the fate and insulinotropic action of L-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na(+) co-transport in the insulinotropic action of L-alanine. In the present study conducted in islets prepared from normal adult rats, L-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-14C]palmitate, (iii) to stimulate 45Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or L-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of L-[U-14C]alanine was unaffected by D-glucose, but inhibited by L-leucine. Inversely, L-alanine decreased the oxidation of D-[U-14C]glucose, but failed to affect L-[U-14C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of L-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na(+) co-transport in the secretory response to L-alanine cannot be ignored.  相似文献   

19.
Modulation of rat brain insulin receptor kinase activity in diabetes   总被引:4,自引:0,他引:4  
Insulin receptors from rat brain regions were studied for insulin binding and receptor associated kinase activity, in alloxan induced short-term and long-term diabetes, and insulin induced hypoglycemia. Insulin receptor activity was assessed by [123I]insulin binding, and basal as well as insulin stimulated kinase activity of the receptor, expressed as phosphorylation of the synthetic peptide poly (Glu-Tyr (4:1)). Regional distribution pattern elicited the highest binding and kinase activity in the olfactory bulb. Diabetes caused a significant increase in the kinase activity. The data suggests that brain insulin receptor kinase is regulated differently compared to peripheral tissues and supports the concept of an active brain insulin receptor in vivo.  相似文献   

20.
Chen H  Nystrom FH  Dong LQ  Li Y  Song S  Liu F  Quon MJ 《Biochemistry》2001,40(39):11851-11859
Phosphoinositide-dependent kinase-1 (PDK-1) is a serine-threonine kinase downstream from PI 3-kinase that phosphorylates and activates other important kinases such as Akt that are essential for cell survival and metabolism. Previous reports have suggested that PDK-1 has constitutive catalytic activity that is not regulated by stimulation of cells with growth factors. We now show that insulin stimulation of NIH-3T3(IR) cells or rat adipose cells may significantly increase the intrinsic catalytic activity of PDK-1. Insulin treatment of NIH-3T3(IR) fibroblasts overexpressing PDK-1 increased both phosphorylation of recombinant PDK-1 in intact cells and PDK-1 kinase activity in an immune-complex kinase assay. Insulin stimulation of rat adipose cells also increased catalytic activity of endogenous PDK-1 immunoprecipitated from the cells. Both insulin-stimulated phosphorylation and activity of PDK-1 were inhibited by wortmannin and reversed by treatment with the phosphatase PP-2A. A mutant PDK-1 with a disrupted PH domain (W538L) did not undergo phosphorylation or demonstrate increased kinase activity in response to insulin stimulation. Similarly, a PDK-1 phosphorylation site point mutant (S244A) had no increase in kinase activity in response to insulin stimulation. Thus, the insulin-stimulated increase in PDK-1 catalytic activity may involve PI 3-kinase- and phosphorylation-dependent mechanisms. We conclude that the basal constitutive catalytic activity of PDK-1 in NIH-3T3(IR) cells and rat adipose cells can be significantly increased upon insulin stimulation.  相似文献   

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