Michio Ui

A new micromethod for the colorimetric determination of inorganic phosphate.

Abstract A sensitive and simple technique was worked out for the colorimetric determination of inorganic phosphate, on the basis of the principle that Malachite Green at a lower pH forms a complex with phosphomolybdate with a marked shift of the absorption maximum. Inorganic phosphate as low as 0.05 μg can be determined by this method with good precision.

An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue

Abstract Taking advantage of the reports of Cailla et al. that succinylation of cyclic AMP and cyclic GMP causes a tremendous increase in their affinities for the antisera (8,9), a simple and ultrasensitive radioimmunoassay has been developed for the simultaneous determination of the cyclic nucleotides in a small volume of biological materials. The binding of succinylated cyclic nucleotides by their specific antibodies is so stabilized in an imidazole buffer that undiluted plasma or the concentrated extract of tissues is directly assayed for cyclic nucleotides without carrying out any prior purification, such as deproteinization of column chromatography. Thus, blood levels of cyclic AMP and cyclic GMP are readily determined in as a small volume of plasma as 50 μl. Periodical changes in the blood concentration of cyclic nucleotides in response to adrenergic and hormonal stimuli were followed in individual rats by means of this method.

Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase

Abstract The guanine nucleotide binding regulatory proteins (N) play an important role in communication between membrane receptors and the adenylate cyclase catalytic unit. One (N s ) of the nucleotide regulatory proteins is involved in the activation, while the other (N i ) is involved in the inhibition of adenylate cyclase. Islet-activating protein (IAP), pertussis toxin, catalyses the transfer of the ADP-ribose moiety of NAD to the active subunit ( M r = 41 00) of N u resulting in a complete loss of the N i functions. IAP will be useful as a tool for further studies of the transmembrane signal transduction mechanism.

A new GTP-binding protein in brain tissues serving as the specific substrate of islet-activating protein, pertussis toxin

A new GTP‐binding protein serving as the specific substrate of islet‐activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an αβγ‐heterotrimeric structure. The α‐subunit of the purified protein (α40βγ) had a molecular mass of 40 kDa and differed from that of Gi(α41βγ) or Go(α39βγ) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross‐reactivities among the three α were different from one another. However, the βγ‐subunit resolved from the three IAP substrates similarly inhibited a membrane‐bound adenylate cyclase and their β‐subunits were immunologically indistinguishable from one another. Thus, the α40βγ is a new IAP substrate protein different from Gi or Go, in the α‐subunit only.

Participation of adenosine 5′‐triphosphate in the activation of membrane‐bound guanylate cyclase by the atrial natriuretic factor

The addition of ANF to Percoll‐purified liver plasma membranes produced a slight activation of guanylate cyclase; the ANF‐stimulated cyclase activity was further increased upon the addition of ATP to the enzyme assay mixture. The effect of ATP to potentiate the cyclase activation was concentration‐dependent, required Mg2+ as a divalent cation, and was seen with membranes from various tissues and cells. ATP increased the maximal velocity of the cyclase without a change in the affinity for GTP or ANF. Phosphorylation by ATP might not be involved since ANF‐stimulated guanylate cyclase was enhanced by non‐phosphorylating ATP analogues as well. Thus, an allosteric ATP binding site is suggested to participate in ANF‐induced regulation of membrane‐bound guanylate cyclase.

A role of phosphofructokinase in pH-dependent regulation of glycolysis

Abstract 1. 1. Glycolysis in a cell-free extract from rat diaphragm was stimulated at elevated pH in the presence of higher concentrations of ATP. A similar situation was also observed in the case of intact-cell preparations of rat diaphragm. 2. 2. It was found that the rate of inhibition of phosphofructokinase (ATP: D -fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) activity by excess ATP was strictly dependent upon the pH; at pH 7.3 an increase in ATP concentration from 1 to 1.7 mM resulted in a sudden inhibition of phosphofructokinase activity, whereas at pH 7.6 such an effect was not observed until the ATP level was raised to 2–3 mM (0.05 mM fructose 6-phosphate was employed as substrate). Consequently phosphofructokinase activity was profoundly influenced by quite a minute change in pH when larger amounts of ATP were present in the glycolytic system. 3. 3. Since the glucose 6-phosphate which accumulated during phosphofructokinase inhibition was inhibitory to hexokinase (ATP: D -hexose 6-phosphotransferase, EC 2.7.1.1), the pH-induced stimulation of phosphofructokinase caused an exaggerated acceleration of overall glycolysis. 4. 4. Preincubation of the cell-free extract without substrates (aging) rendered phosphofructokinase protein unresponsive both to the stimulation on raising pH and to the inhibition by ATP. It is tentatively proposed that the affinity of the inhibitory site(s) of phosphofructokinase for ATP is affected by hydrogen ion concentration and that aging causes a change of conformation of the enzyme, thus blocking the control mechanism of phosphofructokinase.

Islet‐activating protein, pertussis toxin, inhibits Ca2+‐induced and guanine nucleotide‐dependent releases of histamine and arachidonic acid from rat mast cells

Islet‐activating protein Guanine nucleotide‐binding protein Ca2+ Histamine secretion Arachidonic acid

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Abstract: Continuous treatment (1–10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the β‐adrenergic receptor‐coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol‐stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of β‐adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl‐5′‐yl‐β‐γ‐imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p‐induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug‐treated rats. The lag was shortened by the addition of isoproterenol, indicating that β–receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of β‐adrenergic receptor‐mediated adenylate cyclase in the cerebral cortex during desipramine therapy.

Conversion of adrenergic regulation of glycogen phosphorylase and synthase from an α to a β type during primary culture of rat hepatocytes

Abstract Adrenergic regulation of glycogen phosphorylase and synthase was studied with adult rat hepatocytes either immediately after isolation (fresh hepatocytes) or after 24-h maintenance in culture (cultured hepatocytes). In fresh hepatocytes, an α-adrenergic agonist caused stronger activation of phosphorylase than a β agonist, and the effect of epinephrine to activate phosphorylase and to inactivate synthase was suppressed by an α antagonist more efficiently than by a β antagonist. In cultured hepatocytes, however, the relative activities of α- and β adrenergic agents were reversed; a β agonist was much more effective than an α-agonist in activating phosphorylase, and the action of epinephrine on phosphorylase, synthase, and cyclic AMP generation was almost totally blocked by a β antagonist but not by an α antagonist. Such a reciprocal change in hepatic α- and β-adrenergic responses occurred progressively during culture; the change was interfered with by cycloheximide, an inhibitor of protein synthesis, added to the culture medium. Thus, β-adrenergic functions became predominant over α functions when hepatocytes were maintained in primary culture. Physiological significance of this phenomenon is discussed.

Increases in plasma cyclic AMP dependent on endogenous catecholamines

Administration of tyramine (with or without phentolamine) as well as induction of ether anesthesia or insulin hypoglycemia caused a sharp increase in plasma cyclic AMP in rats. Based on the findings that the treatment of rats with reserpine, 6-hydroxydopamine, cocaine or propranolol totally abolished tyramine-induced increases in plasma cyclic AMP, it was concluded that catecholamines released from sympathetic neuronal terminals by tyramine could activate adenylate cyclase via the stimulation of postsynaptic beta-adrenoceptors. In contrast, catecholamines secreted from adrenal medulla were largely responsible for the increase in plasma cyclic AMP induced by ether anesthesia; whereas glucagon, in addition to adrenal catecholamines, played a significant role in hypoglycemia-induced increases in plasma cyclic AMP. Assay of plasma cyclic AMP following these stimuli is very promising as a test for adrenergic activities in experimental and clinical studies.