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1.
2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.  相似文献   

2.
Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.  相似文献   

3.
The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.  相似文献   

4.
The binding of cob(II)alamin (CblII) and 5'-deoxyadenosine to diol dehydrase was studied spectroscopically and with [U-14C]5'-deoxyadenosine. CblII was bound to this enzyme forming a tight 1:1 complex which was resistant to oxidation by O2 even in the presence of CN-. An irreversible 1:1:1 ternary complex was formed between enzyme, CblII, and 5'-deoxyadenosine, when the enzyme was incubated first with the nucleoside and then with CblII. When this order of addition of the constituents was reversed, no 5'-deoxyadenosine was bound to the enzyme-CblII complex. Hydroxocobalamin could also bind to the enzyme together with the nucleoside, while other cob(III)alamins bearing a bulkier Co beta ligand displaced the nucleoside upon binding to the enzyme. The binding of [U-14C]5'-deoxyadenosine was strongly inhibited by unlabeled 5'-deoxy-ara-adenosine, 4',5'-anhydroadenosine, adenosine, adenine, and 5',8-cyclic adenosine, in this order, but not by 5'-deoxyuridine. These results constitute direct evidence for the presence of the binding site for the adenosyl group of adenosylcobalamin, which is spatially limited to and highly specific for adenine nucleosides. The binding of 5'-deoxyadenosine to the apoenzyme was reversible.  相似文献   

5.
Three mutants of Escherichia coli B which are defective in components of the transport system for uridine and uracil were isolated and utilized to study the mechanism of uridine transport. Mutant U- was isolated from a culture resistant to 77 micronM 5-fluorouracil. Mutant U-UR-, isolated from a culture of mutant U-, is resistant to 770 micronM 5-fluorouracil and 750 micronM adenosine. Mutant NUC- is resistant to 80 micronM showdomycin and has been reported previously. The characteristics of uridine transport by E. coli B and the mutants provide data supporting the following conclusions. The transport of adenosine, deoxyadenosine, guanosine, deoxyguanosine, adenine, or guanine by mutant U- and mutant U-UR- is identical with that in the parental strain. Uridine is transported by E. coli B as intact uridine. In addition, extracellular uridine is also rapidly cleaved to uracil and the ribose moiety. The latter is transported into the cells, whereas uracil appears in the medium and is transported by a separate uracil transport system. The entry of the ribose moiety of uridine is fast relative to the uracil and uridine transport processes. The Km values and the inhibitory effects of heterologous nucleosides for the transport of uridine and the ribose moiety of uridine are similar. Studies of cytidine uptake in the parental and mutant strains provide evidence that cytidine is transported by two independent systems, one of which is the same as that involved in the transport of intact uridine. Uridine inhibits but is not transported by the other system for cytidine transport. Evidence for the above conclusions was based on comparisons of the characteristics of [2-14C]uridine, [U-14C]uridine, and [2-14C]cytidine transport using E. coli B and the three transport mutants under conditions which measure initial rates. The nature of the inhibitory effects of heterologous nucleosides on the uridine transport processes and identification of extracellular components from radioactive uridine provides supportive data for the conclusions.  相似文献   

6.
1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.  相似文献   

7.
The pentose cycle and insulin release in mouse pancreatic islets   总被引:35,自引:17,他引:18  
1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.  相似文献   

8.
The pathways of glycogen synthesis from glucose were studied using double-isotope procedures in 18-day cultured foetal-rat hepatocytes in which glycogenesis is strongly stimulated by insulin. When the medium containing 4 mM-glucose was supplemented with [2-3H,U-14C]glucose or [3-3H,U-14C]glucose, the ratios of 3H/14C in glycogen relative to that in glucose were 0.23 +/- 0.04 (n = 6) and 0.63 +/- 0.09 (n = 8) respectively after 2 h. This indicates that more than 75% of glucose was first metabolized to fructose 6-phosphate, whereas 40% reached the step of the triose phosphates prior to incorporation into glycogen. The stimulatory effect of 10 nM-insulin on glycogenesis (4-fold) was accompanied by a significant increase in the (3H/14C in glycogen)/(3H/14C in glucose) ratio with 3H in the C-2 position (0.29 +/- 0.05, n = 6, P less than 0.001) or in the C-3 position (0.68 +/- 0.09, n = 8, P less than 0.01) of glucose, whereas the effect of a 12 mM-glucose load (3.5-fold) did not alter these ratios. Fructose (4 mM) displaced [U-14C]glucose during labelling of glycogen in the presence and absence of insulin by 50 and 20% respectively, and produced under both conditions a similar increase (45%) in the (3H/14C in glycogen)/(3H/14C in glucose) ratio when 3H was in the C-2 position. 3-Mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis from lactate/pyruvate, further decreased the already poor labelling of glycogen from [U-14C]alanine, whereas it increased both glycogen content and incorporation of label from [U-14C]serine and [U-14C]glucose with no effect on the relative 3H/14C ratios in glycogen and glucose with 3H in the C-3 position of glucose. These results indicate that an alternative pathway in addition to direct glucose incorporation is involved in glycogen synthesis in cultured foetal hepatocytes, but that insulin preferentially favours the classical direct route. The alternative foetal pathway does not require gluconeogenesis from pyruvate-derived metabolites, contrary to the situation in the adult liver.  相似文献   

9.
Intestinal preparations from rainbow trout fed a diet rich in wax esters incorporated [1(-14)C]hexadecanoic acid and [1(-14)C]hexadecanol into triacylglycerols at the same rate. The ratio of the number of H atoms from C1 of hexadecanol to the number of molecules of hexadecanol incorporated into triacylglycerols was 1.6 : 3.0. [U-14C]Glucose was incorporated much faster into the glycerol moiety of triacylglycerols than was [U-14C]aspartic acid. We conclude that the oxidation of absorbed fatty alcohol to fatty acid and its subsequent incorporation into triacylglycerols is closely linked with the reductive formation of triacylglycerol-glycerol from glucose. The ability of trout intestines to metabolise fatty alcohol to triacylglycerols was the same in fish fed wax esters as in those fed triacylglycerols.  相似文献   

10.
1. The metabolism and metabolic effects of 3-phenylpyruvate were examined in rat pancreatic islets. 2. Islet homogenates catalysed transamination reactions between 3-phenylpyruvate and L-glutamate, L-leucine, L-norleucine or L-valine. 3-Phenylpyruvate failed to activate glutamate dehydrogenase. 3. 3-Phenylpyruvate rapidly entered into islet cells, was extensively converted into phenylalanine but slowly oxidized. 4. The conversion of phenylpyruvate into phenylalanine coincided with a fall in the content of several amino acids (especially glutamate and aspartate) in the islets and incubation medium, the accumulation of 2-oxoglutarate and a modest fall in the NH4+ production rate. 5. 3-Phenylpyruvate failed to affect 14CO2 output from islets prelabelled with [U-14C]palmitate, but augmented 14CO2 output from islets prelabelled or incubated with L-[U-14C]glutamine. 6. In the presence of L-glutamine, 3-phenylpyruvate augmented the ATP/ADP ratio and NAD(P)H islet content, and caused a rapid and sustained decrease in the outflow of radioactivity from islets prelabelled with [2-3H]adenosine. 7. These data support the view that the insulin-releasing capacity of 3-phenylpyruvate coincides with an increase in the catabolism of endogenous amino acids acting as 'partners' in transamination reactions leading to the conversion of 3-phenylpyruvate into phenylalanine.  相似文献   

11.
Purified rat liver lysosomes ('tritosomes') were prepared from rats injected with Triton WR-1339. 2. The water space of tritosomes, measured by using [3H]water and [14C]sucrose, was 2.15 +/- 0.72 microliter/mg of protein (mean +/- S.E.M., n = 12). 3. Tritosomes, when compared with a crude preparation of normal lysosomes by an indirect method of study, showed sugar specificity but decreased stereospecificity of sugar uptake. 4. At 125 mM the relative rates of net uptake of D-[14C]ribose, D-[14C]- or D-[3H]glucose and 2-deoxy-D-[3H]glucose were the same as that inferred from the indirect study. 5. The entry of D-[3H]glucose into tritosomes showed concentration-dependence suggestive of saturation, with a Km of 48 +/- 18 mM (4). 6. D- and L-glucose, D-ribose, 2-deoxy-D-glucose and D-mannose competed with D-[14C]glucose or D-[14C]ribose for uptake. 7. Cytochalasin B inhibited D-[3H]glucose uptake. 8. Uptake of 1 mM-L-[14C]glucose was slower than for 1 mM-D-[14C]glucose. 9. It is concluded that a facilitated-diffusion transport system is present in purified rat liver lysosomes.  相似文献   

12.
Cell-free, dialyzed extracts from Azotobacter vinelandii rapidly dephosphorylate [U-14C]ATP to labeled ADP and AMP, which is then degraded to hypoxanthine, the end product of AMP catabolism under the experimental conditions which were used. The intermediates of the pathway from ATP to hypoxanthine have been identified by thin layer chromatography and quantitated by the 14-C content. The concentrations of intermediates present during the production of hypoxanthine are consistent with AMP nucleosidase being responsible for AMP degradation in these extracts. This result was confirmed in experiments which utilized rabbit antibody prepared against purified AMP nucleosidase. The antibody inhibited AMP nucleosidase activity in cell-free extracts but did not inhibit adenine demanase or adenosine deaminase from the same extracts. In the presence of antibody prepared against purified AMP nucleosidase, the dialyzed extracts showed a marked reduction in the production of hypoxanthine from ATP. Other enzymes which could be responsible theoretically for the conversion of AMP to hypoxanthine were not detected by standard assay procedures. These results are consistent with AMP degradation proceeding by way of AMP nucleosidase to yield adenine and ribose 5-phosphate. The adenine is then converted to hypoxanthine by adenine deaminase. Both of these enzymes were present in sufficient quantities to account for the observed rates of hypoxanthine formation. The rate of hypoxanthine formation decreases during the time course of the [U-14-C]ATP degradation experiments, even though the concentration of AMP remains high. This decrease in the rate of hypoxanthine formation as a function of time is attributed to the decreasing ATP and increasing P0-4 concentrations, since ATP is an activator of AMP nucleosidase and P0-4 is an inhibitor. These observations suggest that the in vivo activity of AMP nucleosidase could also be regulated by changes in the relative ratios of ATP:AMP:P0-4.  相似文献   

13.
By using wild-type and deoxystreptamine-negative mutants of Streptomyces fradiae grown in media containing [6(-3)H]glucose or [U-14C]glucose, and by subsequent hydrolysis of the labelled neomycin produced, neamines labelled with 3H in both rings I and II, but with 14C in ring I only, were prepared. A mixture of these two forms of neamine was converted by deoxystreptamine-negative Streptomyces rimosus forma paromomycinus into neomycin (not paromomycin) with a 30% yield. The3H: 14C ratio in this neomycin was the same as the measured in neamine produced by hydrolysis of the neomycin, and in unused neamine reisolated from the incubation medium. The 3H:14C ratio in the neomycin was not affected by the presence of unlabelled deoxystreptamine during the incubation. The radioactivity in the neomycin was associated with rings I and II only. It is concluded that the added neamine is incorporated into antibiotic intact, without initial hydrolysis, and that the probable first step in the subunit assembly of neomycin is the formation of neamine.  相似文献   

14.
3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.  相似文献   

15.
The hypothesis that there is impaired endogenous synthesis of glucosamine 6-phosphate in Huntington's-chorea fibroblasts was tested by double labelling matched pairs of fibroblasts in culture with carrier-free H3 32PO4 and [U-14C]glucosamine. The [32P]UDP-N-acetyl[14C]glucosamine and [14C]glucosamine 6-[32P]phosphate of the cellular soluble fraction was isolated by charcoal column and paper chromatography. There is no quantitative difference in 32P but a significant difference in 14C in these two sugars in a ratio of approx. 1.5 for Huntington's-chorea fibroblasts compared with normal fibroblasts.  相似文献   

16.
The metabolism of [1-14C]- and [6-14C]glucose, [1-14C]ribose, [1-14C]- and [U-14C]alanine, and [1-14C]- and [5-14C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of 14CO2 produced from [1-14C]glucose to that from [6-14C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of 14CO2 from [1-14C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of 14CO2 production from [1-14C]glucose was almost linear with time of incubation, whereas that of [6-14C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26 degrees C to 34 degrees C had no appreciable effect on the rates or time courses of oxidation of either [1-14C]- or [6-14C]glucose or of [1-14C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of 14CO2 produced from [1-14C]- to [U-14C]alanine and from [1-14C]- to [5-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
Lactate metabolism in the perfused rat hindlimb.   总被引:2,自引:0,他引:2       下载免费PDF全文
M Shiota  S Golden    J Katz 《The Biochemical journal》1984,222(2):281-292
A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.  相似文献   

18.
The conversion of l-[U-(14)C]lysine into carnitine was demonstrated in normal, choline-deficient and lysine-deficient rats. In other experiments in vivo radioactivity from l-[4,5-(3)H]lysine and dl-[6-(14)C]lysine was incorporated into carnitine; however, radioactivity from dl-[1-(14)C]lysine and dl-[2-(14)C]lysine was not incorporated. Administered l-[Me-(14)C]methionine labelled only the 4-N-methyl groups whereas lysine did not label these groups. Therefore lysine must be incorporated into the main carbon chain of carnitine. The methylation of lysine by a methionine source to form 6-N-trimethyl-lysine is postulated as an intermediate step in the biosynthesis of carnitine. Radioactive 4-N-trimethylaminobutyrate (butyrobetaine) was recovered from the urine of lysine-deficient rats injected with [U-(14)C]lysine. This lysine-derived label was incorporated only into the butyrate carbon chain. The specific radioactivity of the trimethylaminobutyrate was 12 times that of carnitine isolated from the urine or carcasses of the same animals. These data further support the idea that the last step in the formation of carnitine from lysine was the hydroxylation of trimethylaminobutyric acid, and are consistent with the following sequence: lysine+methionine --> 6-N-trimethyl-lysine --> --> 4-N-trimethylaminobutyrate --> carnitine.  相似文献   

19.
Treatment of rats with hypoglycaemic doses of hypoglycin has been shown to abolish the relative detritiation of [2-3H,U-14C]glucose [Osmundsen, Billington, Taylor & Sherratt (1978) Biochem. J. 170, 337-342], indicating that both the Cori and the glucose/glucose 6-phosphate cycles were inhibited in vivo. This inhibition was confirmed and, in addition, it was shown that the conversion in vivo of both [14C]lactate and [14C]fructose into glucose was decreased after hypoglycin treatment. These results suggest that hypoglycin poisoning results in the inhibition in vivo of glucose-6-phosphatase activity, which participates in the overall inhibition of gluconeogenesis and hypoglycaemia. Clofibrate feeding apparently protected the rats against the inhibition of the fructose-to-glucose conversion by hypoglycin. However, in isolated hepatocytes prepared from hypoglycin-treated rats, the conversion of [14C]fructose into glucose and the recycling of [2-3H,U-14C]glucose were not different from that in control hepatocytes. This suggests that the inhibition was lost during preparation of the hepatocytes. The direct measurement of glucose-6-phosphatase activity showed that it was inhibited when measured in concentrated, but not dilute, homogenates prepared from hypoglycin-treated rats.  相似文献   

20.
Synaptosomes, prepared from rat cerebral cortex and hippocampus, were preincubated with [methyl-3H]choline. The effect of adenosine, cyclohexyladenosine, N-ethylcarboxamide adenosine, 2'-deoxyadenosine, and oxotremorine on K+-evoked 3H efflux was investigated. High-voltage electrophoretic separation showed that in the presence of physostigmine, the K+-evoked 3H efflux from hippocampal synaptosomes was 90% [3H]acetylcholine and 10% [3H]choline. Adenosine (30 microM) and oxotremorine (100 microM) both decreased [3H]acetylcholine release from hippocampal synaptosomes. The effect was inversely proportional to the KCl concentration and disappeared at a KCl concentration of 50 mM. Cyclohexyladenosine was approximately 3,000 times more active than adenosine, whereas N-ethylcarboxamide adenosine and 2'-deoxyadenosine were inactive. This indicates that A1 adenosine receptors were involved in the inhibitory effect. Caffeine antagonized the adenosine effect, and at a concentration of 100 microM, it stimulated [3H]acetylcholine efflux. The inhibitory effect of oxotremorine was as great in cortical as in hippocampal synaptosomes. In contrast, adenosine was much less active in cortical than in hippocampal synaptosomes. When inhibitory concentrations of adenosine and oxotremorine were added together into the incubation medium, the effect of adenosine on [3H]acetylcholine release was consistently reduced. An interaction between muscarinic and A1 adenosine presynaptic receptors at a common site modulating acetylcholine release can be assumed.  相似文献   

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