Monophosphate-activated Kinase Research Articles

Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate kinase

The development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (PvGK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the PvGK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z’ factor of 0.6. For the best inhibitor, an IC50 curve was generated.

Pretreatment of rats with increased bioavailable berberine attenuates cerebral ischemia-reperfusion injury via down regulation of adenosine-5′monophosphate kinase activity

Berberine (BBR) exhibits multiple beneficial biological effects. However, poor bioavailability of BBR has limited its clinical application. We previously demonstrated that solid dispersion of BBR with sodium caprate (HGSD) remarkably improves its bioavailability. We examined whether this increased bioavailability of BBR could protect the brain from ischemia-reperfusion (IR) induced injury. Rats treated with HGSD, SC and saline for 7 days then subjected to cerebral ischemia reperfusion by middle cerebral artery occlusion for 2h followed 12h reperfusion. Neurological deficit scores, infarct size, SOD, MDA and NO levels were examined. P-AMPK, Bax, cleaved-Caspase-3 in brain was determined. To further probe for the mechanism of beneficial effect of HGSD, PC12 cells were incubated with serum from control or HGSD pretreated animals, incubated with 300μM H2O2 to induce apoptosis. Caspase-3 activity and cell apoptosis was evaluated. HGSD pretreatment significantly attenuated neurological deficit scores, reduced infarct size, increased SOD and decreased MDA and NO after cerebral IR injury compared to controls. Meanwhile, HGSD pretreatment significantly reduced expression of p-AMPK, Bax, cleaved-Caspase-3 after cerebral IR injury. Sodium caprate (100mg/kg/d) pretreatment alone did not exhibit any of these beneficial effects. PC12 cell apoptosis was attenuated when cells were cultured with HGSD serum compared to control. The presence of AMPK activator (AICAR) attenuated whereas AMPK inhibitor (Compound C) augmented the protective effect of HGSD serum on PC12 cell apoptosis.The results indicate that HGSD-pretreatment of rats protects the brain from ischemia-reperfusion injury and the mechanism is due to its anti-apoptotic effect mediated by decreased activation of AMPK.

Puerarin prevents cardiac hypertrophy induced by pressure overload through activation of autophagy

This study aimed to explore the effects of puerarin on autophagy in cardiac hypertrophy. Decreased 5′-adenosine monophosphate kinase (AMPK) activity alone with inhibited autophagy could be detected in rats within 3 weeks after aortic banding (AB). Puerarin treatment for 3 weeks in AB rats significantly restored autophagy. Administration of puerarin for 6 weeks effectively restricted cardiomyocyte hypertrophy and apoptosis. In an in vitro study, similar anti-hypertrophy and anti-apoptosis effects of puerarin on isoprenaline-induced H9c2 cells were also observed. After inhibition of autophagy by pretreatment with 3-methyladenine, the protective effects of puerarin were blocked. Further in vivo study demonstrated that puerarin significantly enabled phosphorylation of 5′-AMPK to be activated, subsequently inhibiting expression of the mammalian target of rapamycin (mTOR) target proteins S6 ribosomal protein and 4E-binding protein 1. All these data indicate that puerarin exerts protective effects against cardiomyocyte hypertrophy and apoptosis, partly by restoration of autophagy through AMPK/mTOR-mediated signaling.

Adenosine monophosphate kinase (AMPK) expression and activity in the lung

AMPK activity has been implicated in endothelial cell death and vascular smooth muscle proliferation in hypoxia, suggesting that this metabolic sensor participates in vascular pathology. However, molecular mechanisms of AMPK signaling in the lung are poorly understood. Initial studies revealed that the 2 isoforms of AMPK (AMPKα1 and AMPKα2) are expressed at low levels in the rat lung but that hypoxia induces AMPK expression in an isoform‐ and location‐dependent manner. AMPKα1 expression is predominantly induced in the lung periphery and is associated with capillaries while AMPKα2 is expressed in larger vessels. Selective expression of AMPK isoforms extends to distinct pulmonary cell types: pulmonary artery endothelial cells (PAECs) express AMPKα2 while pulmonary microvascular endothelial cells (PMVECs) express AMPKα1. Smooth muscle cells (SMCs) and columnar epithelia express both isoforms. Studies using PAEC, PMVEC, and SMC cultures revealed that AMPK inhibition increases SMC growth and attenuates PMVEC growth. Immunochemical staining indicated that AMPK α1 co‐localizes with the cell adhesion molecule N‐cadherin near regions of cell‐cell contact in PMVECs, results confirmed in co‐immunoprecipitation assays. In conclusion, AMPK expression and function in the lung exhibits both isoform and cell‐type specificity. Supported by research and training grants from NIH.

Regulation of cAMP on the first mitotic cell cycle of mouse embryos

Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo.

Polyphosphate:AMP Phosphotransferase as a Polyphosphate-Dependent Nucleoside Monophosphate Kinase in Acinetobacter johnsonii 210A

We have cloned the gene for polyphosphate:AMP phosphotransferase (PAP), the enzyme that catalyzes phosphorylation of AMP to ADP at the expense of polyphosphate [poly(P)] in Acinetobacter johnsonii 210A. A genomic DNA library was constructed in Escherichia coli, and crude lysates of about 6,000 clones were screened for PAP activity. PAP activity was evaluated by measuring ATP produced by the coupled reactions of PAP and purified E. coli poly(P) kinases (PPKs). In this coupled reaction, PAP produces ADP from poly(P) and AMP, and the resulting ADP is converted to ATP by PPK. The isolated pap gene (1,428 bp) encodes a protein of 475 amino acids with a molecular mass of 55.8 kDa. The C-terminal region of PAP is highly homologous with PPK2 homologs isolated from Pseudomonas aeruginosa PAO1. Two putative phosphate-binding motifs (P-loops) were also identified. The purified PAP enzyme had not only strong PAP activity but also poly(P)-dependent nucleoside monophosphate kinase activity, by which it converted ribonucleoside monophosphates and deoxyribonucleoside monophosphates to ribonucleoside diphosphates and deoxyribonucleoside diphosphates, respectively. The activity for AMP was about 10 times greater than that for GMP and 770 and about 1,100 times greater than that for UMP and CMP.

Pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase deficiency.

Leptin, and Lipid Oxidation Through AMPK

One of the serious effects of diabetes is the excess storage of fat not only in adipocytes, but also in other tissues (termed lipotoxicity). Type II diabetes patients are insensitive to treatment with insulin, and it is hypothesized that insulin resistance results from an excessive accumulation of lipids in nonadipocytes such that the lipids interfere with the action of insulin on those cells. The hormone leptin, which can regulate food intake and neuroendocrine activity, now appears to regulate lipid oxidation in the mitochondria of nonadipocytes. Minokoshi et al. found that leptin injection in mouse muscle in vivo resulted in increased intracellular concentrations of adenosine 5′-monophosphate (AMP), and activated the α2 adenosine 5′-monophosphate-activated kinase (AMPK)--a protein that regulates cellular energy metabolism--in mouse muscle. However, not only did leptin activate α2 AMPK immediately in muscle, it also indirectly caused long-term activation of AMPK (for several hours) as mediated through the hypothalamus. The hypothalamus-dependent activation of AMPK was mediated by the α-adrenergic pathway. Leptin-mediated activation of AMPK led to the phosphorylation (and inactivation) of acetyl coenzyme A carboxylase (ACC), an enzyme that inhibits the import of lipids into and the oxidation of lipids in the mitochondria, and led to increased lipid oxidation in vivo. Leptin failed to increase phosphorylation of ACC in cells that overexpressed a dominant-negative form of AMPK, suggesting that leptin acts through AMPK to inhibit ACC and increase lipid oxidation. Thus, designing therapeutic drugs that target the leptin-AMPK pathway may prove useful for some diabetic patients. Y. Minokoshi, Y.-B. Kim, O. D. Peroni, L. G. D. Fryer, C. Müller, D. Carling, B. B. Kahn, Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415 , 339-343 (2002). [Online Journal] J. Friedman, Fat in all the wrong places. Nature 415 , 268-269 (2002). [Online Journal]

Human herpesvirus 8 open reading frame 21 is a thymidine and thymidylate kinase of narrow substrate specificity that efficiently phosphorylates zidovudine but not ganciclovir.

Human herpesvirus 8 (HHV8) open reading frame (ORF) 21 is predicted to encode a protein similar to the thymidine kinase (TK) enzyme of other herpesviruses. Expressed in mammalian cells, ORF 21 was found to have low TK activity, based on poor growth in media containing hypoxanthine-aminopterin-thymidine (HAT) and low incorporation of [(3)H]thymidine into high-molecular-weight DNA. Kinetic analysis using HHV8 TK as a purified glutathione S-transferase (GST) fusion protein showed that the enzyme has a comparatively high K(m) for thymidine (dThd) of approximately 33.2 microM. Nearly 50% of the phosphorylated product of the reaction with dThd was thymidylate. This monophosphate kinase activity was more pronounced with 3'-azido-3'-deoxythymidine (AZT), in which 78% of the reaction product was AZT diphosphate. Thymidine analogs competitively inhibited dThd phosphorylation by HHV8 TK, while 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, and corresponding analogs did not. Further competition experiments revealed that the nucleoside analog ganciclovir (GCV), at up to 1,000-fold molar excess, could not significantly inhibit dThd phosphorylation by the enzyme. In support of these data, 143B TK(-) cells expressing HHV8 TK phosphorylated GCV very poorly and were not susceptible to GCV toxicity compared to parental cells. Phosphorylation of [(3)H]GCV by a purified GST-HHV8 TK fusion protein was not detected by high-pressure liquid chromatography analysis. Structural features of HHV8 TK substrate recognition were investigated. Therapeutic implications of these findings are discussed.

Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth.

Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.

Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cells.

The signaling pathways mediating relaxation by vasoactive intestinal peptide (VIP), peptide histidine-isoleucine amide (PHI), isoproterenol (ISO), and sodium nitroprusside (SNP) were examined in dispersed rabbit and guinea pig gastric muscle cells. In rabbit muscle cells, SNP stimulated only guanosine 3',5'-cyclic monophosphate (cGMP) and cGMP-dependent protein kinase (cG-kinase) activity; VIP stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP, and both cG-kinase and cAMP-dependent protein kinase (cA-kinase) activities; PHI and ISO stimulated only cAMP and cA-kinase activity, and at higher concentrations, cross-activated cG-kinase. All four agents elicited concentration-dependent relaxation. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1 microM) selectively inhibited cA-kinase activity and abolished relaxation when only cA-kinase was elevated. 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy- 1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c,d,e)- trinden-1-one (KT-5823; 1 microM) selectively inhibited cG-kinase activity and abolished relaxation when only cG-kinase was elevated. When both kinases were elevated, H-89 and KT-5823 partially inhibited relaxation and abolished relaxation in combination. In permeabilized guinea pig and rabbit muscle cells, all agents elicited relaxation and inhibited inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. Both functions were inhibited in parallel fashion by protein kinase inhibitor PKI(6-22) and by KT-5823. We conclude that cA-kinase and cG-kinase act separately and in concert to inhibit IP3-dependent Ca2+ release and induce relaxation.

Improved Spectrophotometric Assay of Nucleoside Monophosphate Kinase Activity Using the Pyruvate Kinase/Lactate Dehydrogenase Coupling System

Improved Spectrophotometric Assay of Nucleoside Monophosphate Kinase Activity Using the Pyruvate Kinase/Lactate Dehydrogenase Coupling System

Decreased Pyrimidine Nucleoside Monophosphate Kinase Activity in Sickle Erythrocytes

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin-sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5'-nucleotidase (P5N), 6-phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin-sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Decreased pyrimidine nucleoside monophosphate kinase activity in sickle erythrocytes

We have previously shown that physiologic concentrations of hemin cause marked inhibition of several red blood cell (RBC) enzymes. Because endogenous heme content is elevated in sickle RBCs, we have examined the activity of hemin-sensitive enzymes in these RBCs. One of the hemin- sensitive enzymes, pyrimidine nucleoside monophosphate kinase (PNMK), was shown to have decreased activity in sickle RBCs relative to RBCs of equivalent cell age. The other hemin-sensitive enzymes, including adenylate kinase (AK), pyrimidine 5′-nucleotidase (P5N), 6- phosphogluconate dehydrogenase (6PGD), and aldolase, had activities that were appropriate for cell age. We have also examined the affinity of the hemin-sensitive enzymes to hemin. Using two different methods, PNMK was shown to have the highest binding affinity to hemin. The exquisite sensitivity of PNMK to inhibition by hemin, coupled with the enzyme's high affinity to hemin, may account for the decrease in PNMK activity and the lack of significant decrease in the other hemin- sensitive enzymes in sickle RBCs. These results suggest that the increased endogenous heme content in sickle RBCs may be responsible for the decrease in PNMK activity. Whether the increased endogenous heme content of sickle RBCs can cause hemolysis indirectly by inhibiting RBC enzymes remains to be determined.

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A 2 and activation by a 70 kDa protein

Plasma membranes were isolated from carrot ( Daucus carota L.) cells grown in suspension culture and treated with phospholipase A 2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A 2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A 2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the V max but does not affect the K m of the phospholipase A 2-released PI kinase.

Molecular characterization of Saccharomyces cerevisiae URA6 gene. DNA sequence, mutagenesis analysis, and cell cycle regulation relevant to its suppression mechanism to cdc8 mutation.

Yeast SOC8 DNA fragment was isolated as a wild type dominant suppressor of cdc8 mutation. We have used Bal31 deletion analysis to define the minimal 1 kilobase HpaI-NcoI DNA element required for complementing the cdc8 mutation. The complementing sequence harbored a multicopy plasmid also enhanced by uridine monophosphate kinase in crude extracts. DNA sequence analysis revealed an open reading frame encoding a protein with a molecular weight of 24,949. Since our SOC8 sequence was identical to that of URA6 gene, which encodes uridine monophosphate kinase, we conclude that SOC8 is allelic with URA6, and we use the term URA6 hereafter. Northern blotting experiments showed that the size of mRNA is about 0.9 kibobases. Primer extension experiments showed multiple transcriptional starting sites primarily located at -160 The size and the deduced amino acid composition are consistent with information obtained from purified uridine monophosphate kinase. Thus, both molecular genetic and biochemical evidence supports a notion that the URA6 is SOC8 encoding a yeast uridine monophosphate kinase. Mutagenesis analysis of its putative nucleotide-binding site, altering essential lysine to glutamic acid, resulted in loss of its uridine monophosphate kinase activity. Complementation analysis studies indicated that the mutated ura6 gene abolished its ability to complement ura6 mutant cells; nor could it suppress cdc8 mutation. Unlike CDC8, the mRNA level of the URA6 gene did not fluctuate throughout the cell cycle; presumably, the temporal order of these two enzymatic activities might be different during cell cycle progression. These data may explain an incomplete suppression of cdc8 by URA6, as previously observed. Taken together, the results support our previous speculation that the suppression of the cdc8 mutation mechanisms by URA6 is due to the provision of the trans-acting dTMP kinase activity to complement the cdc8 defect.

Rapid light-induced changes in phosphoinositide kinases and H(+)-ATPase in plasma membrane of sunflower hypocotyls.

Irradiation of sunflower (Helianthus annuus L. cv. Russian Mammoth) hypocotyls with white light resulted in a 51% decrease in plasma membrane phosphatidylinositol monophosphate (PIP) kinase activity. As little as 10 s of white light irradiation was sufficient to lower the phosphatidylinositol bisphosphate (PIP2) produced in the in vitro phosphorylation assay. This decrease was not caused by an increase in phospholipase C activity since analysis of the water-soluble products indicated no increase in inositol bisphosphate or inositol trisphosphate. Treatment of the plasma membrane with 200 microM vanadate prior to phosphorylation enhanced the PIP kinase and appeared to overcome the light inhibition. In addition to decreasing the PIP kinase activity, light irradiation resulted in a corresponding decrease in the H(+)-ATPase activity to 53% of the dark control values. The plasma membrane ATPase activity increased approximately 2-fold when PIP or PIP2 was added to the isolated membranes. Thus, effects of external stimuli on the level of plasma membrane PIP or PIP2 could affect plasma membrane ATPase activity directly and thereby provide an alternative mechanism for control of cell growth.

Ketone body and phosphoenolpyruvate formation by isolated hepatic mitochondria from Squalus acanthias (spiny dogfish)

AbstractIn elasmobranchs, triglycerides are stored in the liver, which appears to be the primary site for lipolysis and oxidation of fatty acids, a major product being ketone bodies. The objective of this study was to obtain comparative information about properties of ketone body and phosphoenolpyruvate formation by liver of Squalus acanthias (spiny dogfish, a representative elasmobranch) by using an isolated hepatic mitochondrial system we have previously described for studying respiration and glutamine‐dependent citrulline synthesis. Acetate and palmitoyl‐CoA are stoichiometrically converted to ketone bodies at a rapid rate. The capability of mitochondria to convert short‐chain fatty acids to ketone bodies indicates that the previously reported inability of dogfish mitochondria to utilize short‐chain fatty acids to support respiration is not due to absence of nucleoside monophosphate kinase activity. At least two acyl‐CoA synthetase activities are present in mitochondrial extracts, one specific for acetate and another with specificity for longer‐chain fatty acids. Utilization of palmitoyl‐CoA is carnitine‐dependent and is inhibited by low concentrations of malonyl‐CoA. Pyruvate and alanine are also rapidly metabolized to ketone bodies. Phosphoenolpyruvate is formed at a significant rate from a number of substrates, including in particular aspartate and malate in the presence of α‐ketoglutarate. The osmolytes urea and trimethylamine oxide activate and inhibit, respectively, phosphoenolpyruvate formation; in contrast, these osmolytes inhibit and activate, respectively, ketone body and citrulline formation by isolated mitochondria. The results are consistent with the reported unique functions of dogfish liver in lipid metabolism and indicate that the basic enzymatic processes at the level of mitochondria appear to be analogous to those present in mammalian species.

Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle.

We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12-14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists.

ThiK and thiL loci of Escherichia coli.

Mutants of Escherichia coli K-12 auxotrophic for thiamine phosphates were produced in stepwise fashion from the polyauxotrophic F- strain JC1552, via intermediate production of thiamine auxotrophs that had lost the enzymatic activity of either phosphomethylpyrimidine kinase or thiamine phosphate pyrophosphorylase. They include two types: one responds to thiamine monophosphate or thiamine pyrophosphate, and the other responds to thiamine pyrophosphate only; the former lacks thiamine kinase activity, and the latter lacks thiamine monophosphate kinase activity, in addition to the enzymatic defects caused by the first mutations. We found two genes, for which we propose the designations thiK and thiL, which govern the activities of thiamine kinase and thiamine monophosphate kinase, respectively. By conjugation and P1 transduction, the thiK locus was mapped at about 25 min, between pyrC and purB and close to fabD. The relative order of thiK with respect to nearby genes was tentatively established as pyrC-ptsG-fabD-thiK-purB. In the case of thiL, the locus was situated at about 9 min, between tsx and acrA and probably 0.2 min clockwise from the former.