Active Ca2+ transport by membrane vesicles from pigeon erythrocytes Stimulation by amino acids,ATP, GTP,P1 and some other cell constituents

Pretreatment of pigeon erythrocyte membrane vesicles with amino acids, ATP, GTP, P_i and some other simple cell constituents (singly and in combination) causes an increase in ATP-dependent Ca²⁺-uptake activity of vesicles upon subsequent incubation with ⁴⁵Ca²⁺ after removal of the above agents from the ‘i’ face. Amino acids augment the stimulation by all stimulatory agents and are required for stimulation by P_i. The effects of amino acids, ATP, GTP and P_i all occur at physiological concentrations. Many if not all of the effects of the mixture of amino acids that occur naturally in the cells can be accounted for by the group transported by the ‘ASC’ transport system of Christensen (Christensen H.N. (1975) Biological Transport, 2nd edn., W.A. Benjamin, Inc., Reading, MA), but not by any single amino acid at its physiological concentration. The effects of ATP and GTP are not mimicked by their non-hydrolysable β, γ-imido analogues nor by the corresponding 3′, 5′-cyclic monophosphates. None of the effects described appears to involve calmodulin. We suggest that amino acid transport plays a role in metabolic regulation through effects on cell [Ca²⁺]. Analogous effects on cell [Ca²⁺] may be involved in the action of the many hormones which augment amino acid accumulation by the ‘A’ amino acid transport system.     

Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

Activation of turkey erythrocyte adenylate cyclase and blocking of the catecholamine-stimulated GTPase by guanosine 5'-(gamma-thio) triphosphate.

By SDS-polyacrylamide gel electrophoresis, mitochondrial proteins having covalently-bound flavin were analyzed. Mitochondria were prepared from the liver of rat injected with radioactive riboflavin. Radioactivity was found to be associated with four protein components. Their subunit molecular weights were 91,000, 72,000, 60,000 and 44,000. The first two components exhibited yellowish fluorescence on a gel under ultraviolet illumination. The component of the highest molecular weight seems to be a new protein containing covalently-bound flavin.     

Guanosine 5', alpha-beta-methylene, triphosphate, a novel GTP analog, causes persistent activation of adenylate cyclase: evidence against pyrophosphorylation mechanism.

To test the hypothesis that guanine nucleotides activate adenylate cyclase by a covalent mechanism involving pyrophosphorylation of the enzyme, we studied the effect of a novel GTP analog, guanosine 5′, α-β-methylene triphosphate (Gp(CH₂)pp), with a methylene bond in the α-β-position that is stable to enzymatic hydrolysis. Gp(CH₂)pp was as effective as GTP in stimulating rat reticulocyte adenylate cyclase in the presence of isoproterenol. Previously only guanine nucleotides with modified terminal phosphates such as guanylyl 5′-imidodiphosphate (Gpp(NH)p) were thought capable of causing persistent activation of adenylate cyclase. Gp(CH₂)pp, however, caused persistent activation of rat reticulocyte and turkey erythrocyte adenylate cyclase. We conclude that guanine nucleotides do not activate adenylate cyclase by a pyrophosphorylation mechanism and that a modified γ-phosphate is not essential in guanine nucleotides for generation of the irreversibly-activated enzyme state.     

Isolation of homologous and heterologous complexes between catalytic and regulatory components of adenylate cyclase by forskolin-Sepharose

Homologous and heterologous complexes between catalytic and GTP-binding components can be isolated by means of immobilized succinyldeacetylforskolin (forskolin-Sepharose). A heterologous complex is formed by reconstitution of forskolin-Sepharose bound catalytic function from rabbit myocardial membranes with the homogenous [3H]methyl-GTP-binding protein from duck erythrocyte membranes. Analysis of the reconstituted complex by sodium dodecyl sulfate polyacrylamide gelelectrophoresis reveals that only the Mr 42 000 component of the GTP-binding protein's Mr 42 000/Mr 35 000 heterodimer contributes to the formation of active adenylate cyclase.     

Catecholamine binding to the α-adrenergic receptors of hamster adipocytes evidence that guanine nucleotides regulate this binding to the α2-receptor subtype

The binding characteristics of the α-component of (?)-[³H]norepinephrine to hamster adipocyte membranes were studied. Binding was rapid, reaching equilibrium in 20 min at 25°C. Dissociation of specific binding by 10 μM phentolamine suggested dissociation from two different sites. The time course of dissociation induced by a 50-fold dilution was unchanged by the addition of norepinephrine, suggesting the absence of cooperative binding sites. [³H]norepinephrine binding was saturable, yielding curvilinear Scatchard plots. Computer modeling of these data further supported the existence of two classes of binding sites, one with high affinity (_D = 23 nM) but low binding capacity (96 fmol/mg protein) and one with low affinity (K_D = 400 nM) but high binding capacity (1000 fmol/mg protein). Adrenergic ligands of competed with [³H]norepinephrine binding in the following order of potency: (?)-norepinephrine>(?)-epinephrine>>(+)-norepinephrine>(?)-isoproterenol. Displacement by the selective α-adrenergic drugs prazosin, clonidine and yohimbine yielded biphasic curves consistent with binding of [³H]norepinephrine to both α₁- (14–22%) and α₂- (78–86%) receptor subtypes. Although Gpp(NH)p failed to alter the binding of [³H]dihydroergocryptine, it severely reduced the binding affinity of (?)-epinephrine, (?)-norepinephrine and the selective α₂-agonist, clonidine. The inhibitory effects of clonidine and of the α-component of (?)-epinephrine on the adrenocorticotropin-stimulated cyclic AMP production in the intact adipocyte were closely correlated with their effects on the binding of both [³H]norepinephrine and [³H]dihydroergocryptine. Conversely, yohimbine but not prazosin markedly antagonised the α-inhibitory effect of norepinephrine on cyclic AMP production. These data led to concluded that [³H]norepinephrine can be successfully used to study the entire α-adrenergic receptor population of hamster fat cells and that the predominant α₂ -receptor subtype exists in two different affinity states for agonists, the proportions of which are modulated by guanine nucleotides.     

Specific photoaffinity crosslinking of [125I]cholecystokinin to pancreatic plasma membranes. Evidence for a disulfide-linked Mr 76 000 peptide in cholecystokinin receptors

In rat pancreatic plasma membranes, preincubated with [125I]cholecystokinin-33 (CCK-33) and washed free of unbound tracer, the irradiation by UV light induced the irreversible binding of radioactivity to high molecular weight peptides as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and autoradiography. This was not observed when the membranes were preincubated in the simultaneous presence of [125I]CCK-33 and of either an excess of unlabelled CCK-8 or of guanosine 5'-(beta, gamma-imido)-triphosphate. The radioactivity was mostly crosslinked with a Mr 96,000 peptide and peptide species of Mr greater than 200,000, after SDS solubilization in the absence of beta-mercaptoethanol. Peptide reduction with beta-mercaptoethanol converted the high molecular weight radioactive species into a Mr 76,000 peptide that contained as much as 65% of the radioactivity crosslinked. The Mr 76,000 peptide appears, therefore, to be a disulfide-linked constituent of rat pancreatic cholecystokinin receptors.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation

Various analogs of adenosine 5′-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles.It is shown that the fluorophosphate analog ATP(γF) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(γF) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions.The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and P_i. In contrast to ATP(γF), it is a strong inhibitor of both soluble and membrane-bound mitochondrial ATPases.The biological implication of the complementary effects of ATP(γF) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans I. Inhibition by heparin of gonadotropin- stimulated ovarian adenylate cyclase

Heparin inhibits (I₅₀ = 2 μg/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5′-(β,γ-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by hepatin (I₅₀ = 6 μg/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Herapin (3 μg/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged herapin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Guanine nucleotides protect adenylate cyclase against inhibition by Pb2+

The inhibition of rat liver adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Pb²⁺ could be separated into an irreversible and a reversible component.Evidence was obtained that both types of inhibition were due to free Pb²⁺, rather than Pb/ATP, and that Pb²⁺ did not act via the site wherein Mg²⁺ and Mn²⁺ activate the cyclase.Guanine nucleotides strongly counteracted the reversible inhibition of cyclase by Pb²⁺, providing onother example of guanine nucleotide effects on adenylate cyclase function.It is suggested that the Pb²⁺-inhibited cyclase may be of value in the study of guanine nucleotide-cyclase interactions.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.