Responsibility of tRNA(Ile) for spermine stimulation of rat liver Ile-tRNA formation

To determine whether tRNA or aminoacyl-tRNA synthetase is responsible for spermine stimulation of rat liver Ile-tRNA formation, homologous and heterologous Ile-tRNA formations were carried out with Escherichia coli and rat liver tRNA(Ile) and their respective purified Ile-tRNA synthetases. Spermine stimulation was observed only when tRNA from the rat liver was used. Spermine bound to rat liver tRNA(Ile) but not to the purified aminoacyl-tRNA synthetase complex. Kinetic analysis of Ile-tRNA formation revealed that spermine increased the Vmax and Km values for rat liver tRNA(Ile). The Km value for ATP and isoleucine did not change significantly in the presence of spermine. Furthermore, higher concentrations of rat liver tRNA(Ile) tended to inhibit Ile-tRNA formation if spermine was absent. Spermine restored isoleucine-dependent PPi-ATP exchange in the presence of rat liver tRNA(Ile), an inhibitor of this exchange. The nucleotide sequence of rat liver tRNA(Ile) was determined and compared with that of E. coli tRNA(Ile). Differences in nucleotide sequences of the two tRNAs(Ile) were observed mainly in the acceptor and anticodon stems. Limited ribonuclease V1 digestion of the 3'-32P-labeled rat liver tRNA(Ile) showed that both the anticodon and acceptor stems were structurally changed by spermine, and that the structural change by spermine was different from that by Mg2+. The influence of spermine on the ribonuclease V1 digestion of E. coli tRNA(Ile) was different from that of rat liver tRNA(Ile). The results suggest that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation.     

Increase in fidelity of rat liver Ile-tRNA formation by both spermine and the aminoacyl-tRNA synthetase complex

To examine the polyamine effects on the fidelity at the aminoacylation level and the physiological significance of the existence of the aminoacyl-tRNA synthetase complex (ARSC) in animal cells, a single-chain Ile-tRNA synthetase (IRSS) was isolated from the complex by treatment with trypsin. Ile-tRNA formation by IRSS was strongly stimulated by spermine, similar to the results with ARSC. Two misacylations (Val-tRNAIle and Ile-tRNAiMet formation) by IRSS were measured. The error frequency was higher in Ile-tRNAiMet formation (tRNA misacylation) than in Val-tRNAIle formation (amino acid misacylation). Spermine did not influence significantly Ile-tRNAiMet formation, but it stimulated Val-tRNAIle formation by IRSS. Accordingly, spermine decreased the error frequency of tRNA misacylation, but not amino acid misacylation. These results suggest that the conformational changes of individual tRNA by spermine differ from each other, meaning that spermine influences the interaction between individual tRNA and aminoacyl-tRNA synthetase variously. When the aminoacylations of tRNAIle from rat liver, yeast, and Escherichia coli were compared with ARSC and IRSS, the relative speed of Ile-tRNA formation with tRNAIle from other species was faster with IRSS than with ARSC. This indicates that ARSC can recognize tRNAIle from the same species more specifically than IRSS. These results show that both spermine and ARSC are involved in the increase of fidelity of rat liver Ile-tRNA formation.     

The effects of adrenal and gonadal steroids and K+-canrenoate on the metabolism of aldosterone by rat liver microsomes

The synthesis of polar aldosterone metabolites by rat liver microsomes at physiological concentrations of aldosterone (21.5 nM), was markedly inhibited by progesterone, testosterone, corticosterone, K+-canrenoate and estradiol-17 beta. In contrast, corticosterone and estradiol-17 beta significantly increased the synthesis of reduced aldosterone metabolites by 8- and 15-fold respectively, the majority of which were 5 alpha-reduced products of aldosterone. In experiments at higher substrate (aldosterone) concentrations (20-200 microM) the synthesis of ring A-reduced aldosterone metabolites by liver microsomes followed Michaelis-Menten kinetics with a Km[app] for aldosterone of 160 microM and Vmax[app] of 12.2 nmoles/mg protein/5 min. In these experiments progesterone, testosterone and K+-canrenoate all competitively inhibited the synthesis of reduced metabolites with inhibition constants (Ki [app]) of 70, 85 and 55 microM respectively; however, corticosterone did not. In contrast, estradiol-17 beta increased the rate of synthesis of reduced products by 40%, lowering the Km[app] to 83 microM.     

Fetal lamb 3 beta, 20 alpha-hydroxysteroid oxidoreductase: dual activity at the same active site examined by affinity labeling with 16 alpha-(bromo[2'-14C]acetoxy)progesterone

3 beta,20 alpha-Hydroxysteroid oxidoreductase was purified to homogeneity from fetal lamb erythrocytes. The Mr 35,000 enzyme utilizes NADPH and reduces progesterone to 4-pregnen-20 alpha-ol-3-one [Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1] and 5 alpha-dihydrotestosterone to 5 alpha-androstane-3 beta, 17 beta-diol [Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1]. 5 alpha-Dihydrotestosterone competitively inhibits (Ki = 102 microM) 20 alpha-reductase activity, suggesting that both substrates may be reduced at the same active site. 16 alpha-(Bromoacetoxy)progesterone competitively inhibits 3 beta- and 20 alpha-reductase activities and also causes time-dependent and irreversible losses of both 3 beta-reductase and 20 alpha-reductase activities with the same pseudo-first order kinetic t1/2 value of 75 min. Progesterone and 5 alpha-dihydrotestosterone protect the enzyme against loss of the two reductase activities presumably by competing with the affinity alkylating steroid for the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase. 16 alpha-(Bromo[2'-14C]acetoxy) progesterone radiolabels the active site of 3 beta,20 alpha-hydroxysteroid oxidoreductase wherein 1 mol of steroid completely inactivates 1 mol of enzyme with complete loss of both reductase activities. Hydrolysis of the 14C-labeled enzyme with 6 N HCl at 110 degrees C and analysis of the amino acid hydrolysate identified predominantly N pi-(carboxy[2'-14C]methyl)histidine [His(pi-CM)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between spermine stimulation of rat liver Ile-tRNA formation and structural change of the acceptor stem by spermine

We have recently reported that the interaction of spermine with the acceptor and anticodon stems may be important for spermine stimulation of rat liver Ile-tRNA formation [Peng, Z. et al. (1990) Arch. Biochem. Biophys. 279, 138-145]. To pinpoint which interaction of spermine is more important for spermine stimulation of Ile-tRNA formation, Ile-tRNA formation and ribonuclease V1 sensitivity of tRNA(Ile) were studied using purified tRNAs(Ile) from rat liver, wheat germ, brewer's yeast, torula yeast and Escherichia coli. The results indicate that spermine stimulation of rat liver Ile-tRNA formation correlated with the structural change of the acceptor stem by spermine. The nucleotide sequence of wheat germ tRNA(Ile) was also determined.     

Isoleucyl-tRNA synthetase from baker's yeast. Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine

The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.     

Inhibition in vitro of lipogenic enzymes from bovine (Bos taurus) mammary tissue by methylmalonyl-coenzyme A and coenzyme A

Bovine mammary fatty acid synthetase was inhibited by approximately 50% by 40 microM methylmalonyl-CoA; this inhibition was competitive with respect to malonyl-CoA (apparent Ki = 11 microM). Similarly, 6.25 microM coenzyme A inhibited the synthetase by 35% and this inhibition was again competitive (apparent Ki = 1.7 microM). Apparent Km for malonyl-CoA was 29 microM. The short-chain dicarboxylic acids malonic, methylmalonic and ethylmalonic at high concentrations (160-320 microM) and ATP (5 mM) enhanced the synthetase activity by about 50% respectively; the activating effects of methylmalonic acid and ATP on the synthetase were additive. Methylmalonyl-CoA at 50 microM concentration inhibited the partially purified acetyl-CoA carboxylase uncompetitively by 10% and the propionyl-CoA carboxylase activity of the enzyme preparation competitively (apparent Ki = 21 microM) by 40%. Malonyl-CoA also inhibited the acetyl-CoA carboxylase activity competitively (apparent Ki = 7 microM) by 35% and the propionyl-CoA carboxylating activity of the preparation competitively (apparent Ki = 4 microM) by 82%. The possibility that methylmalonyl-CoA may be a causal factor in the aetiology of the low milk-fat syndrome in high yielding dairy cows is discussed.     

Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver

A steady-state kinetic analysis with evaluation of product inhibition was accomplished with purified rat liver flavokinase and FAD synthetase. For flavokinase, Km values were calculated as approximately 11 microM for riboflavin and 3.7 microM for ATP. Ki values were calculated for FMN as 6 microM against riboflavin and for ZnADP as 120 microM against riboflavin and 23 microM against ZnATP. From the inhibition pattern, the flavokinase reaction followed an ordered bi bi mechanism in which riboflavin binds first followed by ATP; ADP is released first followed by FMN. For FAD synthetase, Km values were calculated as 9.1 microM for FMN and 71 microM for MgATP. Ki values were calculated for FAD as 0.75 microM against FMN and 1.3 microM against MgATP and for pyrophosphate as 66 microM against FMN. The product inhibition pattern suggests the FAD synthetase reaction also followed an ordered bi bi mechanism in which ATP binds to enzyme prior to FMN, and pyrophosphate is released from enzyme before FAD. Comparison of Ki values with physiological concentrations of FMN and FAD suggests that the biosynthesis of FAD is most likely regulated by this coenzyme as product at the stage of the FAD synthetase reaction.     

Long-chain acyl-coenzyme A synthetase from rat brain microsomes. Kinetic studies using [1-14C]docosahexaenoic acid substrate

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexaenoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 degrees C. The apparent Km values for ATP (185 microM), CoA (4.88 microM), MgCl2 (555 microM) and [1-14C]docosahexaenoic acid (26 microM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 degrees C (t1/2:3.3 min) and 37 degrees C (t1/2:26.5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 degrees C with the activation energies of 74 kJ/mol between 0 degrees C and 27 degrees C and 30 kJ/mol between 27 degrees C and 45 degrees C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 microM and 29 microM respectively, with V values of 13 and 46 nmol X min-1 X mg protein-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 degrees C and 28 degrees C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 microM and 19 microM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the competition of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of 5'-nucleotidase of a human pancreatic tumor cell line

5'-Nucleotidase of a human pancreatic tumor cell line (PaTu II) has been purified to homogeneity after extraction with detergent followed by two affinity chromatographic steps. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified 5'-nucleotidase revealed a single polypeptide band of 67 kDa. The Western blotted enzyme can be overlaid with concanavalin A proving its glycoprotein nature. After treatment with endoglycosidase F the deglycosylated 5'-nucleotidase exhibits an apparent molecular mass of 58 kDa. The kinetic properties of the solubilized enzyme have been determined (Km (AMP) of 4.0 microM; Vmax (AMP) = 8.6 muMOL/min.mg). Adenosine 5'-[alpha,beta-methylene]diphosphate is a competitive inhibitor of 5'-nucleotidase, whereas concanavalin A inhibits the enzymatic activity in a non-competitive manner. Polyclonal antibodies against purified 5'-nucleotidase of PaTu II have been produced which inhibit its enzymatic activity. Polyclonal antibodies raised against the enzyme purified from rat liver or bull seminal plasma also recognize 5'-nucleotidase of PaTu II cells, whereas polyclonal and monoclonal antibodies against the enzyme derived from chicken gizzard show no cross-reactivity. 5'-Nucleotidase appears to be concentrated in the plasma membrane of PaTu II cells as judged by cell fractionation and indirect immunofluorescence studies.     

Adenylosuccinate synthetase from Dictyostelium discoideum: effects of hadacidin analogs and binding of [14C]hadacidin

The enzyme adenylosuccinate (sAMP) synthetase has been partially purified from Dictyostelium discoideum using hadacidin-Sepharose 4B affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and gel-filtration HPLC, resulting in a 2600-fold purification. Using a newly developed HPLC procedure to assay activity, it has been found that D. discoideum adenylosuccinate synthetase activity has apparent Km values for the substrates IMP, GTP, and aspartate of 36, 23, and 714 microM, respectively. The analog guanosine-5'-(beta, gamma-imino)triphosphate was found to be an inhibitor of GTP with a Ki of 15 microM, and IMP was competitively inhibited by its analog formycin B monophosphate with a Ki of 80 microM. An analysis of these kinetic data showed a pattern consistent with a fully random terter mechanism. Hadacidin, an analog of aspartate, was an inhibitor of that substrate at 86 microM. Other analogs of hadacidin were synthesized and examined for their effect on the sAMP synthetase activity. Compared to hadacidin, which produced 100% inhibition at 5 mM, it was observed that N-acetyl-N-hydroxyglycine, N-formylglycine, N-acetylglycine, and N-hydroxyglycine all inhibited between 50 and 75%, with N-(thiocarboxy)-L-aspartic anhydride less effective at 27%, and N-benzoylglycine at only 6%. N-Formylsarcosine, N-acetylmethionine, O-methylpyruvate oxime, and hadacidin methylester had no effect at this concentration. The adenylosuccinate synthetase activity was dependent on metal ions with maximum activity being obtained with Mg2+. The ability of the aspartate analog hadacidin to bind to the purified adenylosuccinate synthetase was demonstrated using anion-exchange HPLC and [formyl-14C]hadacidin. The radioactivity coeluted with the adenylosuccinate synthetase and the bound, radiolabeled hadacidin was displaced by excess aspartate.     

A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli

Isoleucyl-tRNA synthetase from Escherichia coli catalyzes the activation of [18O2]isoleucine by adenosine 5'-[(R)-alpha-17O]triphosphate with inversion of configuration at phosphorus. Moreover, isoleucyl-tRNA synthetase does not catalyze positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the absence of isoleucine or in the presence of the competitive inhibitor isoleucinol, which effectively eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved. Together, these observations require that isoleucyl-tRNA synthetase catalyzes the activation of isoleucine by associative "in line" nucleotidyl transfer. The synthesis of adenosine 5'-[(R)-alpha-17O]diphosphate and its conversion to adenosine 5'-[(R)-alpha-17O]triphosphate is described and an explanation provided for the reported differences between the treatment of adenosine 5'-[(S)-alpha-thiodiphosphate] with cyanogen bromide and bromine in [18O]water.     

Editing mechanisms in protein synthesis. Rejection of valine by the isoleucyl-tRNA synthetase.

Although the isoleucyl-tRNA synthetase from Escherichia coli (IRS) does not catalyze the overall mischarging of tRNAIle with valine, it does undergo the first step of the reaction, the formation of an IRS-Val-AMP complex. The addition of tRNAIle to this complex leads to its quantitative hydrolysis and the IRS acts as an ATP pyrophosphate in the presence of valine and tRNAIle (Baldwin, A.N., and Berg, P. (1966), J. Biol. Chem. 241, 839). It is shown that during the ATP pyrophosphatase reaction: (a) IRS forms an IRS-Val-AMP complex; (b) the turnover number of the ATP pyrophosphatase reaction is the same at the rate constant for the transfer of isoleucine from IRS-Ile-AMP to tRNAIle over a wide range of temperature and pH; (c) mischarged Val-tRNAIle is hydrolyzed by IRS with a turnover number of 10 s-1 at pH 7.78 and 25 degrees C, compared with a value of 1.2 s-1 for the transfer of isoleucine from IRS-Ile-AMP to tRNA or for the ATP pyrophosphatase reaction. Although this appears to be consistent with an editing mechanism in which there is a slow transfer of the valine from the IRS-Val-AMP to tRNAIle follwed by the rapid hydrolytic step, as recently found for the rejection of threonine by the valyl-tRNA synthetase, there is an inconsistency. This scheme predicts that on mixing IRS.[14C]Val-AMP with tRNAIle there should be a transient misacylation of the tRNA such that about 10% of the [14C]Val is present as [14C]Val-tRNAIle at the peak. But 0.8% or less is found. This could possibly be caused by the IRS having a higher hydrolytic activity during the mischarging reaction than is measured on mixing the unligated enzyme with performed Val-tRNAIle. Alternatively, a two-stage editing mechanism must be considered in which the majority of the Val-AMP is destroyed before the transfer to tRNA in the major editing step, while the hydrolytic activity of the IRS towards Val-tRNAIle is a second editing step to mop up any mischarged tRNA formed by the Val-AMP escaping the first editing step. It is shown that the "kinetic proofreading" mechanism of Hopfield is not consistent with the experimental data.     

ilvU, a locus in Escherichia coli affecting the derepression of isoleucyl-tRNA synthetase and the RPC-5 chromatographic profiles of tRNAIle and tRNAVal.

A mutation in the ilvU locus of Escherichia coli has led to a complex phenotype that included resistance to thiaisoleucine, a loss of derepressibility of isoleucyl tRNA synthetase, and an alteration of the RPC-5 chromatographic profile of the branched-chain aminoacyl-tRNA's. The alterations were manifest in an increase in the amount of Species 2 of both tRNAIle and tRNAVal at the expense of Species 1. A similar alteration, but independent of (and additive to) that caused by the ilvU mutation, was observed upon limitation of either isoleucine or valine. The shift in profile caused by limitation was also independent of the reduced growth rate or the derepression of the isoleucine and valine biosynthetic enzymes that also result from limitation. During chloramphenicol treatment nearly all tRNAIle and tRNAVal formed appears as species 2. Upon recovery from chloramphenicol, Species 2 of both acceptors are converted to Species 1. It is proposed that the ilvU product not only allows derepression of isoleucyl-tRNA synthetase but also retards the conversion of tRNA2Ile to tRNA1Ile and that of tRNA2Val to tRNA1Val. The mutated ilvU loci abolish the derepression and are more efficient in retarding the conversion.     

Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.     

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amylin increases cyclic AMP formation in L6 myocytes through calcitonin gene-related peptide receptors.

The cellular function of amylin is investigated in L6 myocytes, a rat skeletal muscle cell line. Both rat amylin and human amylin-amide acutely cause a dose-dependent increase in cyclic AMP formation in L6 myocytes. 100 nM amylin stimulates intracellular cyclic AMP concentrations 12-fold, whereas human amylin-amide at this concentration causes only a 2-fold increase. Up to 10 mM human amylin has no effect on cyclic AMP levels. Rat calcitonin gene-related peptide (CGRP) is more potent than amylin, causing a 60-fold increase over basal at 1 nM, with an EC50 value of 0.2 nM. The CGRP receptor antagonist, human CGRP8-37 (hCGRP8-37), completely blocks the stimulatory effect of both rat amylin and human amylin-amide on cyclic AMP production. [125I]CGRP binds specifically to a membrane fraction prepared from L6 [125I]CGRP with a Ki of 0.9 nM, while rat amylin also displaces [125I]CGRP with a Ki of 91 nM. Specific binding of [125I]CGRP to plasma membranes of rat liver and brain is also displaced by rat amylin with Ki values of 35 nM and 37 nM, respectively. In contrast, specific binding of [125I]amylin to numerous cells and tissues, under similar conditions, can not be demonstrated. These results suggest that the cellular effects and physiological actions of amylin may be mediated through receptors for CGRP.     

Effect of Indomethacin on cyclic AMP phosphodiesterase activity in myometrium from pregnant rhesus monkeys.

Our results indicate that indomethacin inhibits cyclic AMP phosphodiesterase in the myometrium of the pregnant rhesus monkey under in vitro as well as in vivo conditions. Kinetic data on extracts of myometrium from pregnant rhesus monkeys indicated two cyclic AMP phosphodiesterase activities. The apparent Km value for the high affinity enzyme averaged 3.9 muM and for the low affinity enzyme 23 muM; the Vmax values averaged 0.56 and 1.4 nmoles cyclic AMP hydrolized per mg protein min-1 respectively. When indomethacin was added to the myometrial extracts, the activity of the high Km phosphodiesterase was competitively inhibited, with an average Ki of 200 muM; the low Km enzyme was noncompetitively inhibited with an average Ki of 110 muM. Experiments on myometrial slices demonstrated that 10 muM indomethsacin potentiated the effect of PGE1 and epinephrine on cyclic AMP levels, presumably by inhibiting the phophodiesterase activity. The uterine relaxing effect of indomethacin is generally attributed to the inhibition of prostaglandin synthetase activity. However, treatment of pregnant rhesus monkeys with therapeutic doses of indomethacin resulted in a significant inhibition of myometrial cyclic AMP phosphodiesterase activity in association with uterine relaxation and prolongation of gestation.     

Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver

A low-Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola-cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE-substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size-exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin-activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin-sensitive phosphodiesterase.