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Adenylate cyclase activity of the homogenate of Ehrlich ascites tumor cells pretreated with catecholamine at 37 degrees C was not stimulated by the addition of the same catecholamine, whereas that of the cells without the pretreatment was stimulated. Such a desensitization was induced hardly at all when the pretreatment was performed at low temperature. The desensitization of adenylate cyclase activity to catecholamine stimulation was prevented by pre-pretreatment of the cells with colchicine prior to the catecholamine pretreatment. The effect of colchicine was dependent on the period of the treatment and concentration of colchicine. Vinblastine had a similar effect, whereas cytochalasin B was without effect. Thus, involvement of microtubules was suggested in the desensitization of the membrane-associated enzyme to external stimulation.

When growth-arrested 3Y1 cells (Fischer rat fibroblasts) were exposed to 3 X 10(-5) M colchicine, they entered S phase after a 12-h lag period which is the same as that in serum-stimulated cells. The expression of genes such as c-fos, c-myc, JE, KC, ornithine decarboxylase, and histone H3, analyzed by Northern blotting, increased in a cell-cycle dependent manner after colchicine treatment. The increased level of mRNAs was much smaller in colchicine-stimulated cells than in serum-stimulated cells, corresponding to the lower frequency of the former cells entering S phase. The course of the prereplicative phase seems to be similar in terms of the expression of cell cycle-dependent genes in cells stimulated with colchicine and in those stimulated with serum.

Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), resulted in the desensitization of adenylate cyclase to the beta-adrenergic agonist stimulation depending on the dose and period of the TPA treatment. Treatment of the reticulocytes with TPA caused approximately 40% reduction in the stimulation by beta-adrenergic agonists of adenylate cyclase activity, whereas the treatment had little effect on the basal activity and the activation by fluoride and guanine nucleotide of the enzyme system. No change in the number of beta-adrenergic receptors was observed after the TPA treatment. Treatment with 1-oleoyl-2-acetyl-glycerol (OAG), an activator of protein kinase C, also caused the desensitization of reticulocyte adenylate cyclase to isoproterenol. On the other hand, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C, prevented the desensitization induced by TPA. These results suggest the involvement of protein kinase C in a process of desensitization of adenylate cyclase system to beta-adrenergic agonists in rat reticulocytes.

Production of superoxide anion (O-2) induced by tetradecanoyl phorbol acetate (TPA) in intact guinea pig polymorphonuclear leukocytes (PMNL) was markedly inhibited by a seleno-organic compound, 2-phenyl-1, 2-benzisoselenazol-3 (2H)-one (Ebselen), with glutathione peroxidase-like activity. The compound almost completely inhibited O-2 production by a particulate fraction prepared from TPA-treated PMNL at a concentration as low as 250 nM.

Rabbit antiserum was prepared against hexokinase isoenzyme type I which was purified from rat brain mitochondria. The antiserum inhibited the activity of the mitochondrial hexokinase type I as well as that of the cytosolic type I enzyme prepared from rat brain, kidney and spleen. It did not, however, inhibit the activity of type II hexokinase from muscle and spleen or that of the type III enzyme from spleen. The results suggest that all hexokinase type I isoenzymes may have a common antigenic site irrespective of their sources, though their responses to a thiol inhibitor are different.

Phosphorylation of proteins was examined in guinea pig polymorphonuclear leukocytes in relation to the effects of membrane-perturbing agents, which stimulate superoxide anion production, and their inhibitors. The phosphorylation was detected by 32P autoradiography after separation by two-dimensional electrophoresis of proteins phosphorylated in 32P-preloaded cells. Though phosphorylation of various proteins was stimulated by each of the membrane-perturbing agents, the stimulation was especially marked in six proteins. Phorbol myristate acetate and digitonin enhanced the phosphorylation of the six proteins, while myristate and concanavalin A increased the phosphorylation of five and three proteins, respectively, out of the six proteins, p-Bromophenacyl bromide, an inhibitor of phospholipase A2, inhibited the stimulatory effect of phorbol myristate acetate on both superoxide anion production and protein phosphorylation. Trifluoperazine, a calmodulin inhibitor, also inhibited the effect of phorbol myristate acetate on both, except for an increase in the phosphorylation of one out of the six proteins. α-Methylmannoside, an inhibitor of concanavalin A binding, inhibited the stimulation of the phosphorylation of the three proteins by concanavalin A. The results indicate that the activation of superoxide anion production by the membrane-perturbing agents in guinea pig polymorphonuclear leukocytes is accompanied by the phosphorylation of, at least some of, these six proteins.

Forskolin-activated adenylate cyclase activity in rat erythrocyte membranes was markedly inhibited by methanol in a concentration dependent manner. On the contrary, no change was observed in the basal cyclase activity in the presence of methanol. The methanol inhibition forskolin-activated cyclase activity was protected by the stimulation of Gs in the presence of NaF in a concentration dependent manner. These data indicate that low- and high-affinity binding sites of forskolin in rat erythrocyte membranes have different sensitivities to methanol.

INHIBITION of protein synthesis decreases the rate of DNA synthesis in both prokaryotes and eukaryotes. In prokaryotes, DNA synthesis is dependent on protein synthesis only for the initiation of replication1. In the case of eukaryotes, reduction of DNA synthesis occurs rapidly after the inhibition of protein synthesis2,3, although the mechanism of the tight coupling between protein and DNA synthesis is unclear. The DNA which is synthesised at a reduced rate in these conditions is basically normal except that the elongation step seems to be modified2,3. Newly replicated DNA synthesised in the presence of cycloheximide seems to be associated with less chromatin protein, because chromatin synthesised in these conditions is more susceptible to digestion by DNases3,4. T antigen of simian virus 40 (SV40) is a product of the A gene of the virus5 and has been shown to have a role in the establishment and maintenance of transformation6–9. There is evidence that T antigen is required for the initiation of viral DNA replication10 and the induction of synthesis of cell DNA11–15. It has also been suggested that T antigen initiates DNA synthesis in transformed cells and thereby serves to retain those cells in the transformed state10,16–18. Recently, T antigen was purified almost to homogeneity, and its microinjection into quiescent mouse cells induced DNA synthesis19. However, the mechanism of action of T antigen in cell DNA synthesis remains to be clarified20. If T antigen acts on cellular DNA synthesis, SV40-transformed cells which contain T antigen may behave differently from other cell lines in their ability to continue DNA synthesis in conditions of protein synthesis inhibition. We report here that DNA synthesis in SV40-transformed cells continued at an almost normal rate while protein synthesis was inhibited by cycloheximide, whereas DNA synthesis in malignant tumour cells without T antigen did not continue in these conditions.

Mitochondrial-bound glycerol kinase in rat brain was examined with reference to factors involved in the binding and significance of the binding in relation to ATP metabolism inside the mitochondria. The mitochondrial-bound glycerol kinase was solubilized with glycerol 3-phosphate or ADP, and the solubilized enzyme was rebound to mitochondria by addition of divalent cations. The rebinding was decreased by the presence of glycerol 3-phosphate, while was increased by glucose 6-phosphate. Positive correlation was found between the formation of glycerol 3-phosphate by mitochondrial-bound glycerol kinase and ATP content in mitochondria in experiments using various concentrations of succinate and ADP. On the other hand, glycerol 3-phosphate formation was inhibited by addition of inhibitors for mitochondria functions, such as oligomycin, dinitrophenol, cyanide, and atractyloside. Furthermore, formation of dihydroxyacetone phosphate from glycerol was proved, indicating the involvement of glycerol kinase in glycerol phosphate shuttle in combination with glycerol phosphate dehydrogenase. These findings are discussed in comparison with those of mitochondrial-bound hexokinase.

Two types of cell-cycle-ts mutants were isolated from Fischer rat cell line, 3Y1, and characterized. Clones in one complementation group, tsJT51 and tsJT341, grew at 34 degrees C in the presence of 10% fetal bovine serum (FBS). When the cells growing at 34 degrees C were transferred to 39.5 degrees C, they were arrested alive in G1/G0 phase in the presence of both FBS and epidermal growth factor (EGF), but died in the presence of one of these growth factors. The cells in the other complementation group, tsJT59, tsJT308, tsJT314 and tsJT349, grew at 34 degrees C in the presence of 10% FBS. When the cells growing at 34 degrees C were transferred to 39.5 degrees C, they were arrested alive in G1/G0 phase in the simultaneous presence of FBS, EGF and insulin, but died quickly if one of these growth factors was lacking. Growth-arrested cells at 39.5 degrees C were viable at least one or two weeks and had a potency to resume growth following the shift-down of temperature. Those are assumed to be ts mutant cells which enter and stay in G1/G0 phase from the cell cycle at the non-permissive temperature only in the presence of appropriate growth factors.

When growth-arrested GC-7 cells, a cell line from African green monkey kidney, are stimulated with 10% calf serum, they enter S phase 14-15 h later. Cytochalasin D at 0.6 micrograms/ml blocks the entrance into S phase, and inhibits, though only partially, the increase in protein synthesis after serum stimulation. Since partial inhibition of protein synthesis by cycloheximide interferes with accumulation of labile proteins and thus blocks the entrance of serum-stimulated cells into S phase, the effects of these two inhibitors are compared. Cytochalasin D at lower concentrations reduced the rate of entry into S phase without affecting the length of the prereplicative phase, whereas cycloheximide extended the prereplicative phase dose dependently without affecting the rate of entry into S phase. Cytochalasin D affected neither individual [35S]methionine-labeled spots on two-dimensional polyacrylamide-gel nor degradation of cellular proteins. These results indicate that cytochalasin D, though it interferes with protein synthesis, blocks prereplicative progression of serum-stimulated GC-7 cells in a different manner than cycloheximide.

Transmembrane signalling function of adenylate cyclase system in the cell membranes is discharged and regulated by interactions of the component proteins; receptor protein (either stimulatory (Rs) or inhibitory (Ri)), guanine nucleotide-binding regulatory trimeric protein (either stimulatory (Gs) or inhibitory (Gi)), and catalytic protein (C) for cyclic AMP production (1,2). GTP and its analogs such as guanosine 5′-(β,γ-imino)triphosphate (GppNHp) and guanosine 5′-(γ-thio)triphosphate, regulate the function of G proteins in the presence of Mg2+ (3,4), while F- does in the presence of Al3+ (5). On the other hand, divalent cations, such as Mg2+ and Mn2+, and forskolin directly activate C (6,7). It is also known that cholera and pertussis toxins affect the functions of Gs and Gi, respectively, through ADP-ribosylation of their α-subunits (8, 9).

When human diploid fibroblasts, TIG-1, reached the end of their proliferative lifespan in vitro , they still could synthesize proteins with molecular weight of about 70 000, 85 000 and 116 000 following the heat shock at 46°C for 10 min as well as young cells did so.

The effect of pentobarbital on the adenylate cyclase system was examined in synaptosomal membranes from rat brain. Pentobarbital inhibited forskolin-stimulated enzyme activity more effectively than the basal and Mn2(+)-stimulated enzyme activities. The degree of inhibition of the enzyme activity by pentobarbital was increased by the presence of forskolin in a concentration-dependent manner. No significant difference is observed in the degree of the inhibition by pentobarbital between the basal and forskolin-stimulated activities in the membranes prepared from the peripheral tissues.

The effect of benzodiazepines on adenylate cyclase system was examined in rat brain. Micromolar concentrations of diazepam inhibited the enzyme activity in synaptic membranes in dose- and time-dependent manners. The inhibitory effect of diazepam was more evident on the enzyme activity in the presence of guanylyl-5′-imidodiphosphate (GppNHp) or NaFAlCl3 than on that in the basal state. In the pertussis toxin-treated membranes, the effect of diazepam in the presence of GppNHp or NaFAlCl3 was markedly suppressed. In addition, other benzodiazepines, such as medazepam, flurazepam, flunitrazepam, and clonazepam, had similar effects to those of diazepam, whereas Ro15-1788, an antagonist of a high affinity receptor in the central nervous system, had no effect on adenylate cyclase activity and did not antagonize the effect of diazepam. These findings indicate that benzodiazepines inhibit rat brain adenylate cyclase activity through the effects on both a low affinity benzodiazepine receptor coupled with the inhibitory GTP-binding regulatory protein (Gi) and catalytic protein.

A ts mutant clone, ts JT60, was isolated from Fisher rat cell line, 3Y1. During the exponential growth at both 34 and 39.5 °C, ts JT60 did not appear as ts mutant cells. However, once entered resting state (G0) under serum deprivation at the confluent state, they could re-enter S phase at 34 °C but could not at 39.5 °C following the stimulation of cells either by the addition of fetal bovine serum or by trypsinization and replating. These and other results suggested that ts JT60 is a G0-specific ts mutant, i.e., the cells have ts defect(s) in the function which is required for the stimulation from the resting state to S phase but not for the progression of the cell cycle in an exponential growth phase.

Treatment of rat reticulocytes with isoproterenol resulted in the heterologous desensitization of adenylate cyclase, causing a reduction in NaF/AlCl3- and guanylyl 5'-imidodiphosphate (Gpp(NH)p)-stimulated activities as well as in activity stimulated by beta-adrenoceptor agonists. Desensitization was also induced by dibutyryl cyclic AMP and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of cyclic AMP-dependent protein kinase (protein kinase A), prevented the isoproterenol-induced desensitization, suggesting the involvement of protein kinase A in the desensitization. Gs in the desensitized cell membrane was activated by treatment with Gpp(NH)p or NaF/AlCl3 in the presence of Mg2+. The activated state, the Gs-C complex, was gradually shifted to the basal state, i.e. the Gs-C complex was dissociated by removal of Mg2+. The rate constant of the dissociation was increased in the desensitized cell membranes (0.074 min-1) as compared with that in the control cell membranes (0.022 min-1). The half life of the Gs-C complex, calculated from the rate constant, was decreased during the process of desensitization. The results indicate that reduction in the stability of the Gs-C complex is related to heterologous desensitization. Agents which increase the level of cyclic AMP in the cell also reduced the stability of the complex. The potency of such an effect was in the following order; isoproterenol approximately NaF/AlCl3 (k = 0.071 min-1) greater than forskolin (0.039 min-1) greater than Mn2+ approximately dibutyryl cyclic AMP (0.030 min-1). The former two activate Gs and form Gs-C complex, while the latter two cause intracellular cyclic AMP accumulation without activation of Gs. It may be deduced from this order that the reduction of the stability depends not only on the increase in the cyclic AMP level but also on the level of Gs-C complex formed.

Summary Hexokinase able to bind to mitochondria was purified to homogeneity from rat brain by two successive DEAE-cellulose chromatographic steps. The enzyme lost only the binding ability with almost undetectable change in molecular weight on mild chymotrypsin digestion. The bindable hexokinase was adsorbed to a Phenyl-Sepharose column and eluted with a Lubrol PX gradient, whereas non-bindable hexokinase and yeast hexokinase were not adsorbed under the similar conditions. These results suggest that mitochondria-bindable hexokinase has a hydrophobic region on its surface, which is responsible for the specific interaction with mitochondria.

Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6 M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn2+. An apparent Km value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 microM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the Km value depended on the forskolin concentration up to 1 microM, above which the value converged on ca. 200 microM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mn2+ suppressed the increase in the Km value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn2+ alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn2+ modifies the change.

Two proteins (Mr 46,000, pI 6.4 and 7.0), the phosphorylation of which was increased by any of the membrane-perturbing agents in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes in our previous study (Okamura, N., Ohashi, S., Nagahisa, N. and Ishibashi, S. (1984) Arch. Biochem. Biophys. 228, 270-277), were also phosphorylated in a cell-free system prepared from the leukocytes. The in vitro phosphorylation of these two proteins was stimulated by the addition of phosphatidylserine in the presence of higher concentrations of Ca2+ (300-500 microM). The phosphorylation was further increased when protein kinase C partially purified from guinea-pig brain was added to the system. At a low concentration of Ca2+ (about 10 microM), stimulation of the phosphorylation was not attained by phosphatidylserine alone but required the addition of diacylglycerol or phorbol myristate acetate. On the other hand, the increase in the phosphorylation was inhibited by H-7, an inhibitor for protein kinase C. These results indicate that protein kinase C is involved in the phosphorylation of the two proteins, which may be related to the superoxide anion production stimulated by various membrane-perturbing agents.

tsJT60, a temperature-sensitive (ts) mutant cell line of Fischer rat, is viable at both permissive (34 degrees C) and non-permissive (39.5 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with fetal bovine serum (FBS) from G0 phase they re-enter S phase at 34 degrees C but not at 39.5 degrees. When tsJT60 cells were transformed with adenovirus (Ad) 5 wild type, they grew well at both temperatures, expressed E1A and E1B genes, and formed colonies in soft agar. When tsJT60 cells were transformed with Ad5 dl313, that lacks E1B gene, the transformed cells grew well at 34 degrees C but failed to form colony in soft agar. They died very soon at 39.5 degrees C. 3Y1 cells (a parental line of tsJT60) transformed with dl313 grew well at both temperatures, although neither expressed E1B gene nor formed colonies in soft agar. The phenotype of being lethal at 39.5 degrees C of dl313-transformed tsJT60 cells was complemented by cell fusion with 3Y1BUr cells (5-BrdU-resistant 3Y1), but not with tsJT60TGr cells (6-thioguanine resistant tsJT60). These results indicate that the lethal phenotype is related to the ts mutation of tsJT60 cells and also to the deletion of E1B gene of Ad5.

The mechanism of insulin action on hexokinase isozymes was examined by the electrophoresis on cellulose acetate membrane. It is demonstrated that insulin, some (receptor) protein and type II may form a complex sensitive to sulfhydryl compounds, which appears as type III in some tissues, or as the abortive one with disappearance of type II in others. On this mechanism insulin was deduced to act to induce the protein for the complex as well as to form this complex as a constituent.

Decrease in Type II and concomitant increase in Type III in the hexokinase isoenzymogram of rat carrageenin granuloma were induced in vitro by the addition of insulin to the incubation medium. The changes in the isoenzymogram observed in vitro were quite similar to those induced by insulin in vivo in the same tissue. Addition of pchloromercuribenzene sulfonic acid, a sulfhydryl inhibitor, to the medium also caused decrease in Type II. On the other hand, Type II was markedly increased by the addition of mercaptoethanol, a sulfhydryl compound, to the isoenzyme solution. The findings were discussed with respect to the mechanism of the effect of insulin, especially on the relation between insulin action and state of sulfhydryl groups in the experimental system.

Sodium tricarballylate (8.5 m.mole/kg.) activates the excretion of radioactive strontium injected and reduces its skeketal accumulation. Under a similar condition, sodium lactate (13.5 m.mole/kg.×4) is ineffective. Mechanism of active excretion caused by the injection of tricarballylate was discussed and suggested to be due to its chelating action. Some side effect of tricarballylate injection was observed but it did not produce severe injury.

Effect of tricarballylate, citraconate, tartrate, lactate, and citrate, which are able to form chelating complex with strontium, on the distribution of parenterally administered radioactive strontium was examined, and it was indicated that all of these organic acids markedly decreased skeletal depostion of radiostrontium and four acids other than citrate markedly increased radiostrontium in the blood serum and soft tissues.