Takeo Asakawa

Inhibition of catalytic unit of adenylate cyclase and activation of GTPase of Ni protein by βγ-subunits of GTP-binding proteins

A protein factor which inhibited adenylate cyclase was purified to apparent homogeneity from rat brain and identified as the βγ‐subunits of the GTP‐binding regulatory proteins of adenylate cyclase. (i) The βγ‐subunits (protein factor) inhibited the partially purified catalytic unit of adenylate cyclase in the presence of an activator, forskolin or the stimulative regulatory protein (Ns), to 60 and 40% of the control, respectively; inhibition of the catalytic unit in the presence of forskolin required no guanine nucleotides. (ii) The subunits enhanced the GTPase activity of the purified α‐subunit of the inhibitory regulatory protein (Niα) 3.8‐fold, (iii) The subunits stimulated ADP‐ribosylation of niα catalyzed by islet‐activating protein (pertussis toxin). ADP‐ribosylation had no effect on the GTPase activity of Niα in the presence of the βγ‐subunits. The results suggest that direct inhibition of the catalytic unit by the βγ‐subunits liberated from Ni is essential for the receptor‐mediated inhibition of adenylate cyclase.

Evidence for the Presence of a GTP‐Dependent Regulatory Component of Adenylate Cyclase in Myelin from Rat Brain

Abstract: A GTP‐dependent regulatory component of adenylate cyclase was found in myelin from rat brain. The fraction solubilized from myelin contained a component that reconstituted guanine nucleotide‐responsive adenylate cyclase activity when combined with the catalytic unit of adenylate cyclase prepared from rat brain. Purified myelin demonstrated little adenylate cyclase activity, even in the presence of F− or Mn2+. The reconstituted activity was dependent on the amount of the solubilized myelin fraction and required the presence of 5′‐guanylylimidodiphosphate, a hydrolysis‐resistant analog of GTP. The elution pattern of the component solubilized from myelin in gel filtration was very similar to that of a GTP‐dependent regulatory component from synaptic plasma membranes. The content of the regulatory component‐like activity in myelin was estimated to be 50–60% of that in synaptic plasma membranes. Cholera toxin ADP‐ribosylated proteins having molecular weights of 48,000, 38,000, 23,000, 20,000 and 15,000 and other minor peptides in myelin, some of which were also present in synaptic plasma membranes. We conclude that myelin contained a GTP‐dependent regulatory component of adenylate cyclase despite the apparent lack of adenylate cylcase activity in myelin.

Effects of nitroglycerin on regional myocardial function in the underperfused canine heart

Summary: Effects of nitroglycerin (3 μg/kg/min i.v.) on regional myocardial contractility during acute coronary stenosis were studied in open-chest dogs using a strain-gauge arch. Stenosis-induced stepwise decreases in coronary perfusion pressure (CPP) at <40 mm Hg correspondingly reduced contractility in the underperfused area and increased the left ventricular end-diastolic pressure (LVEDP). Nitroglycerin caused significant increases in contractility, along with decreases in arterial and left ventricular pressures; at stenosis-induced CPP <30 mm Hg, contractility in the underperfused area fell precipitously below the control, while LVEDP increased. When nitroglycerin infusion under coronary stenosis (CPP of 40 mm Hg) decreased CPP to <30 mm Hg, contractility fell. When CPP >30 mm Hg was maintained, contractility increased and LVEDP decreased. In conclusion, at least in the absence of well-developed collateral circulation, the critical level of CPP was 40 mm Hg for contractility and LVEDP without nitroglycerin, which shifted to 30 mm Hg with the addition of nitroglycerin. Nitroglycerin resulted in a significant increase in plasma catecholamines, and the increase in contractility diminished with propranolol, indicating participation of β-adrenoceptor in the positive inotropic effect of nitroglycerin. However, catecholamines at high concentrations probably further aggravated the impaired cardiac function at CPP <30 mm Hg.

GTPase activity associates with the inhibitory GTP-binding regulatory component of adenylate cyclase purified from rat brain.

The inhibitory regulatory component of adenylate cyclase (Ni) was highly purified from rat brain synaptic membranes. A low K m GTPase activity was always associated with Ni through the purification, and the recovery of GTPase activity correlated well with that of Ni. Purified Ni was hardly ADP‐ribosylated by islet‐activating protein (IAP). A heat‐labile factor in the fraction of the stimulative regulatory component (Ns) restored ADP‐ribosylation and also activated the GTPase about 2‐fold. NaF which was reported to interact with Ni markedly reduced GTPase activity. The purified Ni fraction inhibited adenylate cyclase only in the presence of a heat‐stable factor found in the partially purified regulatory component. GTPase and inhibitory activities were weak in myelin which contained only a small amount of Ni. These findings support the view that GTPase activity is an intrinsic activity of Ni and some factors are necessary for the function of Ni.

Ca2+-Activated, Fatty Acid-Dependent Guanylate Cyclase in Synaptic Plasma Membranes and Its Requirement for Ca2+ and Mg-GTP in the Activation

Cyclic GMP in an intact cell system was reported to increase by various biologically active compounds, including muscarinic cholinergic agonists and excitatory amino acids, and supposed to act as intracellular messenger of the various neurotransmitters and hormones. Guanylate cyclase, a cyclic GMP-generating enzyme, in a cell free system can not be stimulated by agents increasing cyclic GMP contents. This suggests that the cyclase is activated by a substance generated or a process taking place within cells upon hormone stimulation. Thus cellular components such as Ca2+, fatty acids and their metabolites that are intracellularly liberated on the stimulation have been supposed to be possible physiological activators of guanylate cyclase in intact cells.

ADP-ribosylation of myelin basic protein by cholera toxin

Cholera toxin ADP-ribosylates four types of myelin basic proteins (MBPs) of Mr 14,000, 17,500, 19,000 and 22,000 in rat brain myelin. On an analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MBP underwent mono- and multi-(ADP-ribosyl)ation by cholera toxin and thus modified MBP migrated on the gel as several discrete protein bands, the molecular masses of which were apparently larger by 500-2000 daltons than that of the corresponding untreated MBP. On average, 1.1 mol of ADP-ribosyl residue was incorporated into 1 mol of MBP. Four types of purified MBPs were also ADP-ribosylated by cholera toxin dependent on GTP and the protein factor for the ADP-ribosylation. The results show evidence that MBP is one of major and specific substrates of cholera toxin in brain membranes.

Activation of rat brain adenylate cyclase by proteases: involvement of distinct protein components in the activation by α-chymotrypsin and trypsin

Adenylate cyclase in synaptic plasma membranes from rat brain is activated by ?-chymotrypsin or trypsin. These proteases also activate adenylate cyclase reconstituted from the catalytic subunit of adenylate cyclase and the partially purified fraction of the GTP-binding proteins containing both the stimulatory and inhibitory GTP-binding proteins. Properties of the activation of reconstituted adenylate cyclase by the proteases are as follows. (1) The proteases do not directly activate the catalytic subunit. However, the pre-treatment of the partially purified GTP-binding proteins with ?-chymotrypsin (100 ?g/ml) increases the subsequently reconstituted cyclase activity at least 3-fold. Trypsin (10-30 ?g/ml) much more weakly enhances the cyclase activity. (2) ?-Chymotrypsin and trypsin synergistically activate the cyclase. (3) Trypsin but not ?-chymotrypsin no longer activates the cyclase when the purified stimulatory GTP-binding protein (Gs) replaces the partially purified GTP-binding proteins. (4) The stimulatory effects of ?-chymotrypsin and trypsin on the cyclase activity are little or slight unless 5?-guanylylimidodiphosphate (Gpp(NH)p) is present in the reconstitution. (5) The purified ??-subunits of the GTP-binding proteins markedly inhibit adenylate cyclase. This inhibition is nearly completely attenuated by treating the ??-subunits with ?-chymotrypsin (> 10 ?g/ml). (6) Trypsin (1-10 ?g/ml) inactivates the GTPase of the ?-subunit of the inhibitory GTP-binding protein (Gi). This inactivation of the GTPase seems to correlate with the activation of the reconstituted adenylate cyclase by trypsin. We conclude that two distinct protein components are involved in the activation of adenylate cyclase by ?-chymotrypsin and trypsin. One component sensitive to ?-chymotrypsin is probably the ??-subunits of the GTP-binding proteins. The other component sensitive to trypsin may be the ?-subunit of Gi.