Affinity-based capture and identification of protein effectors of the growth regulator ppGpp.

The nucleotide ppGpp is a highly conserved regulatory molecule in bacteria that helps tune growth rate to nutrient availability. Despite decades of study, how ppGpp regulates growth remains poorly understood. Here, we developed and validated a capture-compound mass spectrometry approach that identified >50 putative ppGpp targets in Escherichia coli. These targets control many key cellular processes and include 13 enzymes required for nucleotide synthesis. We demonstrated that ppGpp inhibits the de novo synthesis of all purine nucleotides by directly targeting the enzyme PurF. By solving a structure of PurF bound to ppGpp, we designed a mutation that ablates ppGpp-based regulation, leading to dysregulation of purine-nucleotide synthesis following ppGpp accumulation. Collectively, our results provide new insights into ppGpp-based growth control and a nearly comprehensive set of targets for future exploration. The capture compounds developed should also enable the rapid identification of ppGpp targets in any species, including pathogens.

Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study.

Microtubules display the unique property of dynamic instability characterized by phase changes between growth and shrinkage, even in constant environmental conditions. The phases can be synchronized, leading to bulk oscillations of microtubules. To study the structural basis of dynamic instability we have examined growing, shrinking, and oscillating microtubules by time-resolved cryo-EM. In particular we have addressed three questions which are currently a matter of debate: (a) What is the relationship between microtubules, tubulin subunits, and tubulin oligomers in microtubule dynamics?; (b) How do microtubules shrink? By release of subunits or via oligomers?; and (c) Is there a conformational change at microtubule ends during the transitions from growth to shrinkage and vice versa? The results show that (a) oscillating microtubules coexist with a substantial fraction of oligomers, even at a maximum of microtubule assembly; (b) microtubules disassemble primarily into oligomers; and (c) the ends of growing microtubules have straight protofilaments, shrinking microtubules have protofilaments coiled inside out. This is interpreted as a transition from a tense to a relaxed conformation which could be used to perform work, as suggested by some models of poleward chromosome movement during anaphase.

Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus.

The release of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A (BFA) action, preceding the movement of Golgi membrane into the ER. ATP depletion also causes the reversible redistribution of the 110-kD protein from Golgi membrane into the cytosol, although no Golgi disassembly occurs. To further define the effects of BFA on the association of the 110-kD protein with the Golgi apparatus we have used filter perforation techniques to produce semipermeable cells. All previously observed effects of BFA, including the rapid redistribution of the 110-kD protein and the movement of Golgi membrane into the ER, could be reproduced in the semipermeable cells. The role of guanine nucleotides in this process was investigated using the nonhydrolyzable analogue of GTP, GTP gamma S. Pretreatment of semipermeable cells with GTP gamma S prevented the BFA-induced redistribution of the 110-kD protein from the Golgi apparatus and movement of Golgi membrane into the ER. GTP gamma S could also abrogate the observed release of the 110-kD protein from Golgi membranes which occurred in response to ATP depletion. Additionally, when the 110-kD protein had first been dissociated from Golgi membranes by ATP depletion, GTP gamma S could restore Golgi membrane association of the 110-kD protein, but not if BFA was present. All of these effects observed with GTP gamma S in semipermeable cells could be reproduced in intact cells treated with AlF4-. These results suggest that guanine nucleotides regulate the dynamic association/dissociation of the 110-kD protein with the Golgi apparatus and that BFA perturbs this process by interfering with the association of the 110-kD protein with the Golgi apparatus.

Two distinct central serotonin receptors with different physiological functions.

Two distinct serotonin (5-hydroxytryptamine) receptors designated serotonin 1 and serotonin 2 bind tritium-labeled serotonin and tritium-labeled spiroperidol, respectively. Drug potencies at serotonin 2 sites, but not at serotonin 1 sites, predict their effects on the "serotonin behavioral syndrome," indicating that serotonin 2 sites mediate these behaviors. The limited correlation of drug effects with regulation by guanine nucleotides suggests that serotonin 1 sites might be linked to adenylate cyclase. Drug specificities of serotonin-elicited synaptic inhibition and excitation may reflect serotonin 1 and serotonin 2 receptor interactions, respectively.

A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.

The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.

The activation of adenylate cyclase by guanyl nucleotides in Saccharomyces cerevisiae is controlled by the CDC25 start gene product.

In the thermosensitive cdc25 start mutant of Saccharomyces cerevisiae, the regulation of adenylate cyclase by guanyl nucleotides was rapidly nullified when the enzyme was prepared from nonsynchronized cells shifted to the restrictive temperature. In agreement with previous in vivo complementation studies, this biochemical defect was fully suppressed by the expression of either the whole cloned CDC25 gene or its C-terminal portion. Moreover, membranes prepared from cdc25(Ts) cells grown at the permissive temperature evinced an altered regulation of adenylate cyclase by guanyl nucleotides. These results indicate that the CDC25 protein, together with RAS, is involved in the regulation of adenylate cyclase by guanyl nucleotides and raise the possibility that adenylate cyclase might form a ternary complex with RAS and CDC25.

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Evolution of neurotransmitter receptor systems.

The presence of hormones, neurotransmitters, their receptors and biosynthetic and degradative enzymes is clearly not only associated with the present and the recent past but with the past several hundred million years. Evidence is mounting which indicates substantial conservation of protein structure and function of these receptors and enzymes over these tremendous periods of time. These findings indicate that the evolution and development of the nervous system was not dependent upon the formation of new or better transmitter substances, receptor proteins, transducers and effector proteins but involved better utilization of these highly developed elements in creating advanced and refined circuitry. This is not a new concept; it is one that is now substantiated by increasingly sophisticated studies. In a 1953 article discussing chemical aspects of evolution (Danielli, 1953) Danielli quotes Medawar, "... endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competences." To also quote Danielli, "In terms of comparative biochemistry, one must ask to what extent the evolution of these reactivities, reaction patterns and competences is conditional upon the evolution of methods of synthesis of new proteins, etc., and to what extent the proteins, etc., are always within the synthetic competence of an organism. In the latter case evolution is the history of changing uses of molecules, and not of changing synthetic abilities." (Danielli, 1953). Figure 4 outlines a phylogenetic tree together with an indication of where evidence exists for both the enzymes that determine the biosynthesis and metabolism of the cholinergic and adrenergic transmitters and their specific cholinergic and adrenergic receptors. This figure illustrates a number of important points. For example, the evidence appears to show that the transmitters and their associated enzymes existed for a substantial period before their respective receptor proteins. While the transmitters and enzymes appear to exist in single cellular organisms, there is no solid evidence for the presence of adrenergic or cholinergic receptors until multicellular organisms where the receptors appear to be clearly associated with specific cellular and neuronal communication (Fig. 4). One can only speculate as to the possible role for acetylcholine and the catecholamine in single cell organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of guanine ribonucleotide accumulation on the metabolism and cell cycle of human lymphoid cells.

A deficiency of purine nucleoside phosphorylase activity is associated with marked depletion of T-lymphocytes which is felt to be mediated by accumulation and further metabolism of the purine nucleoside phosphorylase substrate, 2'-deoxyguanosine. Human T-lymphoblasts incubated in the presence of 2'-deoxyguanosine and the purine nucleoside phosphorylase inhibitor 8-aminoguanosine accumulate deoxyguanosine 5'-triphosphate whereas B-lymphoblasts and mature T4+-cell lines accumulate GTP under identical conditions. We have compared the effects of guanine ribo- and deoxyribonucleotide accumulation on the metabolism and cell cycle of the respective cell lines. Deoxyguanosine 5'-triphosphate elevations in T-lymphoblasts are associated with inhibition of [3H]uridine incorporation into DNA and a complete block at the G1-S interface of the cell cycle. In contrast 3- to 5-fold increases in guanosine 5'-triphosphate pools in B-lymphoblasts and mature T-cell lines do not inhibit [3H]uridine incorporation into DNA or RNA but do cause a pronounced slowing in the progression of cells through S phase. B-lymphoblasts deficient in the salvage enzyme hypoxanthine guanine phosphoribosyltransferase do not accumulate guanosine 5'-triphosphate from 2'-deoxyguanosine and progress normally through the cell cycle, demonstrating a requirement for guanine salvage to inhibit cell growth. Guanine ribonucleotide accumulation was also associated with inhibition of de novo purine biosynthesis and a moderate decline in adenine nucleotide pools but not with inhibition of protein synthesis or alterations in basal levels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic guanosine monophosphate. We conclude that the accumulation of guanine ribonucleotides by actively cycling human lymphoid cells is associated with an increase in S-phase cells and inhibition of growth. This effect is distinctly different from that produced by 2'-deoxyguanosine 5'-triphosphate and should be taken into account in pharmacological studies with 2'-deoxyguanosine and its analogues.

NUDT15 R139C-related thiopurine leukocytopenia is mediated by 6-thioguanine nucleotide-independent mechanism in Japanese patients with inflammatory bowel disease.

BACKGROUND: NUDT15 R139C (rs116855232) is a recently identified genetic factor responsible for thiopurine-induced leukocytopenia and hair loss. In this study, we investigated the association of NUDT15 R139C with 6-thioguanine nucleotide (6-TGN) levels and thiopurine-induced leukocytopenia in Japanese patients with inflammatory bowel disease (IBD). METHODS: Two hundred and sixty-four subjects (103 healthy volunteers and 161 IBD patients treated with thiopurines) were enrolled. Genotyping for NUDT15 R139C was performed using Custom TaqMan® SNP genotyping assays. RESULTS: The NUDT15 C/C, C/T, and T/T genotypes were 80.7, 18.2, and 1.1 %, respectively. The allelic frequency was 10.2 %. Among 161 IBD patients, there was no significant difference in 6-TGN levels among the NUDT15 genotypes. Forty-five patients (27.9 %) developed leukocytopenia (WBC <3000/µl), and the C/T and T/T genotypes were significantly associated with the development of leukocytopenia (P = 1.7 × 10(-5)). In these patients, 6-TGN levels were not significantly different between NUDT15 genotypes. NUDT15 R139C was significantly associated with early (<8 weeks) (P = 1.03 × 10(-4)) and late (>8 weeks) leukocytopenia (P = 4.3 × 10(-4)). The decrease in WBC count at 2 and 4 weeks was significantly higher in patients with the C/T or T/T genotypes as compared to the patients with the C/C genotype. All patients with the T/T genotype (n = 2) developed early severe hair loss and severe leukocytopenia (<1000/µl). The logistic regression analysis revealed that NUDT15 R139C was the sole genetic factor responsible for the thiopurine-induced leukocytopenia (P = 0.001). CONCLUSIONS: These results suggest that NUDT15 R139C-related thiopurine-induced leukocytopenia is mediated by a 6-TGN-independent mechanism.

Guanine nucleotides stimulate carboxyl methylation of kidney cytosolic proteins.

We studied the effect of guanine nucleotides on the carboxyl methylation catalyzed by class II protein carboxylmethyltransferases (PCMT). Addition of guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) promoted a time- and concentration-dependent enhancement of protein methylation in the cytosolic fraction isolated from kidney cortex. GTP gamma S affected the kinetics of the methylation reaction, as reflected by alterations of both apparent Km and Vmax of the methyltransferase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-L-homocysteine, a well-known inhibitor of methyltransferase-catalyzed reactions. No GTP gamma S stimulation of methylation was found in cytosolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The methylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on L-isoaspartyl residues and thus methylated by a class II PCMT. These results suggest that class-II-associated protein methylation activity from the soluble fraction of the kidney can be regulated by guanine nucleotides.

Guanine nucleotide regulation of the pertussis and cholera toxin substrates of rat glioma C6 BU1 cells.

Rat glioma C6 BU1 cells contain a pertussis toxin substrate of 40 kDa which does not appear to be identical with Gi,Go or transducin. The GTP analogue, GTP[gamma S], inhibited the rate of pertussis toxin-catalysed ADPribosylation of this protein, while the GDP analogue GDP[beta S] stimulated this reaction. A protein of the same kDa value was ADPribosylated by cholera toxin in the absence of added guanine nucleotides. It is suggested that this 40 kDa protein can be a substrate for both cholera and pertussis toxins under appropriate conditions.

Effects of ppGpp on transcription by DNA-dependent RNA polymerase from Escherichia coli: circular dichroism, absorption and specific transcription studies.

Concrete evidence is presented for conformational changes elicited in RNA polymerase upon binding ppGpp by circular dichroism measurements. In the presence of 100 microM ppGpp, the molar ellipticity of RNA polymerase at 220 nm is reduced by 14% from the initial value of - 11,100 deg X cm2 X dmol-1 at 25 degrees C. In vitro transcription on templates containing the beta-lactamase promoter and colicin E1 promoter on poly[d(A-T)] is inhibited by ppGpp. None of these templates had GC-rich nucleotide sequence near the transcription initiation site, and yet they were influenced by ppGpp. Comparison of the effect on the synthesis of mRNAs for beta-lactamase and colicin E1 and the synthesis of the proteins themselves indicates that the effect of ppGpp is at the level of transcription for the former case and involves coupled transcription-translation for the latter case. Difference absorption, polyacrylamide gel electrophoresis, and nitrocellulose filter-binding studies show that the binding of ppGpp to RNA polymerase does not impair the extent of the interaction between enzyme and DNA. Kinetic studies suggest that ppGpp affects transcription initiation on beta-lactamase promoter. On poly[d(A-T)], ppGpp affects the rate of open complex formation and is a mixed inhibitor with respect to the incorporation of nucleotides. Our results are consistent with the idea that ppGpp acts as a regulator by binding at a site different from the active site and changes the RNA polymerase conformation, causing altered transcriptional behavior on different DNA templates.

RNA synthesis and the accumulation of guanine nucleotides during growth shift down in the blue-green alga Anacystis nidulans.

Shift down of growth rate in the blue-green alga Anacystis nidulans by reduction of the incident light intensity produced a reduction in the rate of stable RNA accumulation which was correlated with increased concentrations of three phosphorylated compounds, two of which were identified as guanosine 5'-diphosphate-3'-diphosphate (ppGpp) and guanosine 5'-triphosphate3'-diphosphate (pppGpp). The step also causes a large but transient increase in the concentration of GTP. Stable RNA fails to accumulate for a considerable length of time after the concentrations of ppGpp and pppGpp have fallen, suggesting the involvement of another mechanism in the control of stable RNA accumulation.

Reduction of mono(ADP-ribosyl)ation of 20 kDa protein with maturation in rat testis: involvement of guanine nucleotides.

When the homogenate prepared from immature rat testes was incubated with [32P]NAD, several proteins (90, 39 and 20 kDa) were ADP-ribosylated in the absence of bacterial toxins. This observation suggested the existence of an endogenous ADP-ribosyltransferase and substrates. The data that the digested product by phosphodiesterase of ADP-ribosylated 20 kDa protein was 5'-AMP suggested that 20 kDa protein was mono(ADP-ribosyl)ated. In addition, the mono(ADP-ribosyl)ation of 20 kDa protein was enhanced by guanine nucleotides such as GTP, GDP and GTP[gamma S], and decreased by the concentrations of 10 mM Mg2+. In contrast, the incorporation of ADP-ribose moiety from NAD to both 90 and 39 kDa proteins was not changed by guanine nucleotides. On the other hand, mono(ADP-ribosyl)ation of 20 kDa protein was not observed in the homogenate prepared from other tissues of the same rats. Furthermore, we found that mono(ADP-ribosyl)ation of 20 kDa protein was decreased with the maturation of the rats and that an endogenous mono(ADP-ribosyl)transferase and 20 kDa protein were located in the nuclei.

Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteins.

We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.

Biphasic effect of sodium fluoride and guanyl nucleotides on binding to prostaglandin E2 receptors in rat epididymal adipocyte membranes.

Both NaCl and NaF promoted PGE2 binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the 'salt effect' of sodium on PGE2 binding, the effects of Mg2+ and guanyl nucleotides on PGE2 binding in the presence of NaCl or NaF were compared. Mg2+ decreased PGE2 binding; high NaF concentration abolished this inhibition, while increased NaCl concentrations did not affect the Mg2+ inhibition. In the presence of Mg2+ the effects of NaCl and NaF were additive. The enhancement of PGE2 binding by fluoride, unlike sodium, was dependent on the presence of Mg2+. Incubation of the membranes with GDP beta S, Gpp(NH)p, GTP or GTP gamma S increased PGE2 binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE2 binding at low NaF concentrations and inhibition of PGE2 binding at high NaF concentrations. No changes in the stimulatory action of NaCl on PGE2 binding were observed in the simultaneous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE2 binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE2 binding. The implications of these findings are discussed.

Adenylate cyclase of osteoblast-like cells from rat osteosarcoma is stimulated by calcitonin as well as parathyroid hormone.

Isolated bone cells are often described according to the presence of PTH- and calcitonin (CT)-sensitive adenylate cyclase activities. Osteoblasts are thought to be cells with PTH-sensitive adenylate cyclase but without CT response, and osteoclasts are thought to be CT-sensitive cells. We have studied the adenylate cyclase of a cloned bone cell line (UMR-106) derived from a rat osteosarcoma and used as a model of osteoblastic cells. Cells maintained in continuous culture for over 2 yr contain adenylate cyclase responsive to CT as well as PTH. The stimulatory effects of both hormones are dependent on hormone concentration, time, and the guanine nucleotide GTP. PTH and CT may activate the same adenylate cyclase in UMR-106 cells, since the stimulatory effects of the two hormones are not additive when combined at concentrations giving maximal activity. The beta-adrenergic agonist isoproterenol also stimulates adenylate cyclase in these cells. Unlike late passages of UMR-106 cells, cells of earlier passages (less than 50) showed only slight CT-sensitive adenylate cyclase activity. Our results suggest that studies of hormone effects attributed to the osteoblast phenotype should consider possible alteration of hormone responsiveness in cloned tumor cells during long term culture.

Properties of gonadotropin-stimulable adenylyl cyclase of the human corpus luteum: regulation of hormonal responsiveness by guanyl nucleotide and magnesium ion.

We studied the properties of a gonadotropin-responsive adenylyl cyclase in membrane preparations obtained from human corpus luteum and explored the nature of guanyl nucleotide and Mg ion involvement in activation of the enzyme. Maximal adenylyl cyclase activity required ATP concentrations of 1-2.5 mM, total MgCl2 concentrations of 8-10 mM, and 1 mM EDTA. Optimal hCG responsiveness, however, required lower (approximately 5 mM) MgCl2 concentrations. Both GTP and its hydrolysis-resistant analog guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] increased enzyme activity, but the response to each nucleotide had distinct characteristics. The rate and the extent of activation were greater in the presence of GMP-P(NH)P than in that of GTP. Moreover, enzyme activation by GMP-P(NH)P was hysteretic in nature, requiring about 8 min to reach steady state velocity in the absence of hormonal stimuli. The slow rate of activation by GMP-P(NH)P was accelerated by either hCG or increases (3-10 mM) in the concentration of MgCl2. Thus, both gonadotropin and Mg ion are inherently antihysteretic in the human luteal adenylyl cyclase system. Basal and hCG stimulation were under the control of guanine nucleotides. Dose-response curves showed that the apparent activation constants for GTP and GMP-P(NH)P were 0.30 and 0.51 microM, respectively; these values did not shift after the addition of hCG. At a higher concentration of guanyl nucleotides (1000 microM), basal and hCG-stimulated activities were markedly reduced, suggesting bimodal regulation of the enzyme by the nucleotides. We also found that enzyme responsiveness to prostaglandin E2 was small and that, in contrast to a number of other nonprimate species, adenylyl cyclase from the human corpus luteum was not stimulated by isoproterenol. Taken together, these data support the usefulness of the cell-free model for studying the role of adenylyl cyclase in the regulation of luteal function in the human.

Calcium-permeable channels activated via guanine nucleotide-dependent mechanism in human carcinoma cells.

Patch clamp experiments on human carcinoma A431 cells have revealed two types of Ca2(+)-permeable channels, the activity of which can be increased by the application of non-hydrolyzable analogues of GTP to the intracellular side of the membrane. With 105 mM Ca2+ in recording pipette at 30-33 degrees C their unitary conductances (in pS) are 1.3 (SG-channels) and 2.4 (G-channels). G- and, possibly, SG-channels are activated from the extracellular side of the membrane with epidermal growth factor (EGF). The data are consistent with the hypothesis that both channels are activated via guanine nucleotide binding (G) proteins.