Reporting negative studies in the mass media.
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Bias↗
Biomedical subjects
J E Bishop
Publications and source records attributed to J E Bishop.
Synthesis and evaluation of high affinity, aryl-substituted [18F]fluoropropylbenzamides for dopamine D-2 receptor studies.
The potent dopamine D-2 ligands (S)-2,3-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5(3- [18F]fluoropropyl)benzamide (18F-1) and (S)-2,3-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(3- [18F]fluoropropyl)-6-hydroxybenzamide (18F-2) were prepared in high specific activity and 5-25% overall radiochemical yields. Benzamide 1 possessed a lower in vitro binding affinity for the D-2 receptor than salicylamide 2, but the in vivo striatal-to-cerebellar radioactivity concentration ratios (St/Cb) in rats and dogs were nearly identical for the two compounds. Compound 18F-2 was more lipophilic than 18F-1, and its increased penetration into and retention by striatal tissue was matched by an increase in non-specific binding in the cerebellum. Cerebral cortex radioactivity concentration levels in dogs were similar to cerebellum levels. The binding of 18F-labelled 1 and 2 displayed regional brain distribution patterns consistent with known dopamine D-2 receptor densities and was selectively blocked in the striatum of rats by dopamine D-2 antagonists. The binding of 18F-1 was found to be stereoselective, as the 18F-labelled (R)-enantiomer displayed no selective retention in the striatum of dogs. High levels of radioactivity were found in the bones of rats following the injection of 18F-1 and 18F-2, indicating that in vivo defluorination had occurred; however, no bone radioactivity was observed in dogs following the injection of these radioligands. Compound 18F-1 was displaced from the striatum of dogs by both d-amphetamine-stimulated dopamine release and haloperidol at doses of 1 and 0.5 mg/kg, respectively, while compound 18F-2 was displaced from the dog striatum only by haloperidol at these doses. The radioligand 18F-2 holds promise for positron emission tomography studies of the dopamine D-2 receptor system based upon its selective, potent binding and resistance to displacement by endogenous dopamine.
Animals↗
1,25(OH)2-vitamin D3, a steroid hormone that produces biologic effects via both genomic and nongenomic pathways.
The hormonally active form of vitamin D is 1,25(OH)2-vitamin D3 [1,25(OH)2D3]. This seco-steroid is the key mediator of the vitamin D endocrine system which produces biological effects in over 28 target tissues. In these target tissues, the biological responses may be generated both by a signal transduction mechanism which involves a nuclear receptor for 1,25(OH)2D3 that modulates gene transcription or a signal transduction pathway which involves rapid opening of Ca2+ channels which are externally located on the plasma membrane. This paper reviews the evidence in support of the pleiotropic effects of this steroid hormone and presents evidence that the receptor of the genomic effects is likely to be separate from the receptor/membrane recognition element which initiates the rapid nongenomic biological effects.
Animals↗
1,25-Dihydroxyvitamin D3 in the Atlantic cod: plasma levels, a plasma binding component, and organ distribution of a high affinity receptor.
Physiological studies of the Atlantic cod, Gadus morhua, have suggested a role for the vitamin D3 system in this marine teleost similar to that in other vertebrates. Accordingly, the present study was carried out to assess the plasma concentrations of vitamin D3, 25-hydroxyvitamin D3 (25OHD3), and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in this fish. Additionally, the presence of binding proteins in plasma and target-specific tissue receptors for these vitamin D3 metabolites was studied in organs normally associated with calcium regulation. Plasma levels of 25-OHD3 (undetectable to 148 pg/ml; n = 5) were comparatively low (20-30 ng/ml), whereas the levels of vitamin D3 (approximately 30 ng/ml) and 1,25-(OH)2D3 (approximately 50 pg/ml) were comparable to levels reported in higher vertebrates. Cod plasma contained a protein that bound both 25OHD3 and 1,25-(OH)2D3. This plasma binding protein revealed low affinity for 25OHD3, did not bind G-actin, and had a sedimentation coefficient of 3.4S. High affinity 1,25-(OH)2D3 receptors [Kd, 1.02 +/- 0.36 (n = 6), 1.02 +/- 0.3 (n = 5), and 0.95 +/- 0.51 (n = 5) nM; mean +/- SEM] were found in high salt cytosols from intestine, liver, and gills, respectively, and had sedimentation coefficients (3.6-3.8S in 0.3 M KCl sucrose gradients) similar to those in higher vertebrates. No specific 1,25-(OH)2D3 binding was found in kidney, ultimobranchial glands, corpuscles of Stannius, or bone. The finding of significant quantities of 1,25-(OH)2D3 in the plasma, the presence of plasma binding proteins that bind this seco-steroid, and the localization of specific high affinity receptors for this metabolite in calcium regulatory tissues in teleosts are all consistent with a physiological role for the vitamin D3 system in the calcium regulation of the cod.
Adrenocorticotropic Hormone↗
Local chromatin changes accompany the expression of the calbindin-D28K gene: tissue specificity and effect of vitamin D activation.
The high affinity calcium-binding protein calbindin-D28K is one of the known proteins transcriptionally up-regulated by the hormonally active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. This regulation is tissue specific, since in the absence of 1,25-(OH)2D3, the expression of calbindin-D28K is virtually abolished in intestine, whereas it is decreased, but clearly detectable, in kidney, and it remains present at its highest level in cerebellum. Several studies have shown that there is a strong correlation between an increase in the sensitivity to nuclease digestion of a given gene locus and its potential for transcription. Furthermore, hypersensitive sites have often been mapped to regions of DNA including or surrounding sequences known to be important for the regulation of gene transcription. In this study we have scanned the 5'-end and flanking DNA of the calbindin-D28K gene for the presence of DNase-I-hypersensitive (DH) sites in order to localize possible regulatory regions involved in the tissue-specific and hormone-dependent regulation of this gene. We have found that in tissues where calbindin is not expressed, such as liver, no DH sites could be detected. In cerebellum, the same set of DH sites was observed in the presence or absence of 1,25-(OH)2D3 treatment, reflecting the vitamin D-independent expression of the calbindin gene in this tissue. A more complex pattern of DH sites was found in intestine, independently of the vitamin D status of the animal.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
1,25-Dihydroxyvitamin D3 receptor RNA: expression in hematopoietic cells.
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes.
Calcitriol↗
Evidence for 1,25-dihydroxyvitamin D3 production by cultured porcine alveolar macrophages.
Previous studies have demonstrated that human alveolar and bone marrow macrophages when activated in vitro can metabolize 25-hydroxyvitamin[3H]D3 to 1 alpha,25-dihydroxyvitamin[3H]D3; however, to date no animal models to study this system have been available. In the present study, cultured porcine pulmonary alveolar macrophages from two animals were assayed for their capability for metabolism of 25-hydroxyvitamin[3H]D3. The porcine alveolar macrophages constitutively produced a metabolite of 25-hydroxyvitamin[3H]D3 which was identified as 1 alpha,25-dihydroxyvitamin[3H]D3 by high performance liquid chromatography. The apparent KM was in the range of 300 nM. Unlike human macrophages, treatment of porcine alveolar macrophages with lipopolysaccharide did not stimulate 1 alpha,25-dihydroxyvitamin[3H]D3 production. Addition of 1 alpha,25-dihydroxyvitamin-D3 to macrophages cultures led to a sensitive proportional inhibition of 1 alpha,25-dihydroxyvitamin[3H]D3 synthesis.
Animals↗
Synthesis and in vitro evaluation of 2,3-dimethoxy-5-(fluoroalkyl)-substituted benzamides: high-affinity ligands for CNS dopamine D2 receptors.
A number of 2,3-dimethoxy-5-(fluoroalkyl)-N-[(1-ethyl-2- pyrrolidinyl)methyl]benzamides (with or without a 6-hydroxy group) were synthesized and evaluated as dopamine D2 receptor ligands. The parent acids were synthesized via the Claisen rearrangement of the appropriate O-allyl ethers, which were derived from o-vanillic acid or 2,3-dimethoxysalicylic acid. A decrease in reactivity was found to be characteristic of pentasubstituted benzoates, and difficulties were encountered with the introduction of fluorine onto the ethyl side chains. The (fluoroethyl)- and (fluoropropyl)salicylamides were 5 times more potent than the corresponding benzamides in inhibiting [3H]spiperone binding to the D2 receptor. These (fluoroalkyl)salicylamides are of potential value for in vivo positron emission tomography (PET) studies upon the basis of their relatively selective, high potency binding affinity for the D2 receptor.
Animals↗
Long-term changes in mouse lung following inhalation of a fibrosis-inducing dose of 239PuO2: changes in collagen synthesis and degradation rates.
Mice were exposed by nose-only inhalation to 239PuO2, which resulted in an IAD of 1110 +/- 29 Bq. At various times after exposure, rates of collagen metabolism were measured using validated in vivo methods based on the administration of radiolabelled proline, together with a large flooding dose of unlabelled proline and measurement of its incorporation into lung collagen as hydroxyproline. Dramatic increases in both synthesis and degradation rates of collagen were observed. At 54 days after exposure the fractional synthesis rates in experimental mice were almost five times those in controls (control: 3.2 +/- 0.6%/day, 239PuO2-exposed: 14.5 +/- 0.4%/day) and by 300 days synthesis rates, although declining, were still more than double the control values. A similar pattern of change was observed for collagen degradation. The combination of changes in synthesis and degradation rates led to a 60% increase in lung collagen content by 300 days (control: 3.05 +/- 0.24 mg/lung, 239PuO2-exposed: 4.88 +/- 0.42 mg/lung). The data suggest that extensive remodelling of the lung connective tissue matrix occurs during development of fibrosis and that, over long periods of time, small imbalances between synthesis and degradation may result in quite large increases in protein content.
Administration, Inhalation↗
Lymphocyte cell lines from vitamin D-dependent rickets type II show functional defects in the 1 alpha,25-dihydroxyvitamin D3 receptor.
Lymphocyte cell lines were established from five patients with vitamin D-dependent rickets, type II (VDDR-II). These lines were established by infection with human T-lymphotrophic virus type I (HTLV-I). Binding of [3H]1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) to its receptor in these cell lines was compared to binding studies using a T-lymphocyte cell line (S-LB1) from a normal individual. The 1,25(OH)2D3 receptor of S-LB1 was comparable to the well-characterized chick intestinal 1,25(OH)2D3 receptor in terms of its ligand binding affinity and capacity, its mobility on 5-20% sucrose gradients, and its adsorption to and elution properties from DNA-cellulose. Three cell lines established from patients with VDDR-II (Rh-VDR, Sh-VDR, and Ab-VDR) showed no specific binding of 1,25(OH)2D3 to a receptor and treatment of the cultured cells with 1,25(OH)2D3 did not stimulate production of 24,25-dihydroxy-vitamin D3 (24,25(OH)2D3), a response which is diagnostic of the presence of a functional 1,25(OH)2D3 receptor. In a fourth cell line, A1-VDR, the receptor for 1,25(OH)2D3 had a low binding capacity and 25(OH)D3-24-hydroxylase activity was not detectable. Induction of 24,25-(OH)2D3 synthesis by 1,25(OH)2D3 was observed in the fifth cell line, designated Ro-VDR, although the sensitivity to hormone treatment was lower than in the control cell line from a normal donor. The capacity of the receptor for 1,25(OH)2D3 was low in Ro-VDR. In all cell lines where 1,25(OH)2D3 binding to a receptor was detectable, the receptor had the typical sedimentation coefficient of 3.7 S on sucrose density gradient analysis. Binding and elution properties to DNA-cellulose, however, differed from normal in both Ro-VDR and A1-VDR cells where elution from DNA-cellulose occurred at a lower salt concentration than is typical of the 1,25(OH)2D3 receptor. While Ro-VDR cells showed typical nuclear localization of the unoccupied 1,25(OH)2D3 receptor, neither the unoccupied nor the occupied receptor from A1-VDR cells was completely localized in the nucleus. In a series of functional studies we found that modulation of the level of the mRNAs coding for both the c-myc oncogene and the growth factor known as granulocyte-monocyte colony stimulating activity by 1,25(OH)2D3 correlated with the 1,25(OH)2D3 receptor status of these cells. Use of these cell lines will facilitate further study of the molecular defect(s) in the receptor for 1,25(OH)2D3 in vitamin D-dependent rickets type II and will allow a correlation with impairment of cellular functions.
24,25-Dihydroxyvitamin D 3↗
Enhanced deposition of predominantly type I collagen in myocardial disease.
The myocardium consists of a muscle fibre array surrounded and interspersed by a network of connective tissue, principally collagen, which maintains the functional integrity of the heart. Changes in collagen composition may therefore contribute to altered ventricular function. Collagen composition was examined in cardiac tissue from 15 patients undergoing orthotopic cardiac transplantation. Of these, 10 had severely impaired left ventricular function due to coronary artery disease. The remaining five had dilated cardiomyopathy. Normal heart tissue was taken at autopsy from 25 patients who died of causes unrelated to cardiovascular disease. Left ventricular collagen concentration, estimated from hydroxyproline levels, increased from 48.6 +/- 4.1 mg/g dry weight of tissue in the control group to 95.3 +/- 9.7 mg/g (P less than 0.01) in patients with dilated cardiomyopathy and to 63.5 +/- 9.8 mg/g in the coronary artery disease group. This increase was attributable to an increase in absolute concentrations of both type I and III collagen, determined by separation of cyanogen bromide peptides by sodium dodecyl sulphate polyacrylamide gel electrophoresis. However, there was a significant decrease in the proportion of type III collagen (compared with type I plus III) from 41.8 +/- 1.1% in controls, to 34.6 +/- 1.5% (P less than 0.01) in the coronary artery disease group and 35.8 +/- 2.8% (P less than 0.05) in the dilated cardiomyopathy group. These results suggest that excessive collagen production, with a preponderance of type I, occurs in these forms of myocardial disease, indicative of a remodelling of the collagen matrix, which, by increasing passive myocardial stiffness may contribute to impaired heart function seen in these groups of patients.
Adult↗
Changes in the composition and metabolism of arterial collagens during the development of pulmonary hypertension in rabbits.
Increased pulmonary artery pressure is known to result in enhanced collagen deposition in the pulmonary artery. Here we investigate how changes in collagen metabolism may bring about this increased deposition in the pulmonary artery of animals with pulmonary hypertension induced by bleomycin. Rabbits were injected intratracheally with bleomycin sulfate or with saline. After 14 days the animals were injected with L-[U-14C]proline plus a "flooding" dose of unlabeled proline. Uptake into arterial collagens and release of labeled hydroxyproline were then measured after 2.5 h. The relative amounts of types I and III collagens were assessed from the levels of cyanogen-bromide-derived peptides alpha 1(I)CB8 and alpha 1(III)CB5, respectively, after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Collagen synthesis rates of about 3%/day were found in the control pulmonary artery and aorta, and about one-half of the newly synthesized collagen was degraded rapidly. Fourteen days after bleomycin, there was a fivefold increase in collagen synthesis rate (p less than 0.01) and a marked decrease in the percentage of newly synthesized collagen degraded rapidly. There was no change in collagen metabolism in the aorta of these animals. Pulmonary artery collagen from control rabbits consisted of 26.5 +/- 1.0% type III collagen. There was no change in composition in bleomycin-treated animals. This study demonstrates quite rapid turnover rates for collagen in normal blood vessels. Our results also indicate that remodeling of arterial connective tissue matrix during pulmonary hypertension involves marked but commensurate increases in type I and III collagens brought about by changes in both synthesis and degradative processes.
Animals↗
A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors.
There is currently much interest in the role of mediators that regulate cell proliferation. Methods to assay proliferative effects of such mediators usually involve cell counting techniques, which are tedious to perform, or methods based on uptake of radiolabelled thymidine, which may be prone to errors caused by precursor pool artefacts. We describe here an assay for estimating the number of adherent cells present in a microculture and its application to the study of growth factors. The assay depends on the binding of Methylene Blue to the fixed monolayer at pH 8.5 and, after washing the monolayer, release of dye by lowering pH. The use of an elution solvent containing acidified ethanol ensures a linear correlation between absorbance of the dye and cell number, and enables the assay to be carried out in 96-well plates measuring absorbance with an automated vertical light-path microplate photometer. The assay is rapid, highly reproducible and easy to perform, making it ideal for screening large numbers of samples. It was shown to be applicable to a number of foetal and adult cell lines derived from man and experimental animals. It was also demonstrated to be useful for assaying purified growth factors and detecting growth promoting activity in cell and tissue extracts.
Animals↗
Age-related changes in the proportion of types I and III collagen.
Previous studies of the age-related changes in interstitial collagens, have suggested that the proportion of type III collagen compared with type I decreases with age. In this study collagen concentration and the proportion of types were measured in heart, lung and skin of male Lewis rats aged between 1 day and 2 years. Collagen concentration, based on hydroxyproline levels, increased in all tissues up until 6 months of age, thereafter it increased in heart and lung, yet decreased in skin. The relative proportions of types I and III collagen were assessed after cyanogen bromide digestion of the tissue and separation of the resultant peptides by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At 2 weeks of age collagen type III represented about one-third of types I and III in all tissues. After this age the proportion of type III increased up to 1 year in both heart (53.9 +/- 1.8%) and lung (47.7 +/- 2.8%), whereas it decreased in skin (18.6 +/- 1.5%). These data show that there are age-related changes in both collagen concentration and the relative proportions of types I and III collagen, but that the direction of these changes differs between tissues.
Aging↗
Exclusive A-ring linkage for singly attached phycocyanobilins and phycoerythrobilins in phycobiliproteins. Absence of singly D-ring-linked bilins.
Previous spectroscopic studies on the phycocyanobilin-containing peptide beta-2T from Synechococcus sp. 6301 C-phycocyanin and the phycoerythrobilin-containing peptide beta-2TP from Porphyridium cruentum B-phycoerythrin indicated a different single thioether mode of attachment, postulated to be through the D-ring of the tetrapyrrole, in contrast to the A-ring linkage established for the other singly linked bilins in these proteins (Bishop, J.E., Lagarias, J.C., Nagy, J. O., Schoenleber, R.W., Rapoport, H., Klotz, A.V., and Glazer, A.N. (1986) J. Biol. Chem. 261, 6790-6796; Klotz, A.V., Glazer, A.N., Bishop, J.E., Nagy, J.O., and Rapoport, H. (1986) J. Biol. Chem. 261, 6797-6805). The crystal structure of Agmenellum quadruplicatum C-phycocyanin at 2.5-A resolution (Schirmer, T., Bode, W., and Huber, R. (1987) J. Mol. Biol., 196, 677-695) supports an A-ring linkage for all three phycocyanobilins. Consequently we have re-evaluated our proposed structural assignments by further 1H NMR studies. Two-dimensional homonuclear correlated and nuclear Overhauser enhancement spectroscopic data presented here show that all three bilins in Synechococcus 6301 C-phycocyanin are attached solely through the A-ring, complementary to the crystallographic data. The evidence from the NMR data for all bilin peptides examined includes the dipoledipole interactions of the 5-H with the 3-H, 3'-H, and a pyrrole methyl group (7-CH3); the corresponding interactions would not be possible in a D-ring-linked bilin. The 5-H also consistently exhibits allylic J-coupling to the 3-H, supporting A-ring linkage assignment. These data are inconsistent with the alternative D-ring linkage assignment since this would involve J-coupling through five bonds. Examination of the phycoerythrobilin beta-2 position in B-phycoerythrin also reveals an A-ring type of attachment by similar criteria. We conclude that all singly linked bilins are attached through the A-ring.
Chemical Phenomena↗
(Iodoacetamido)fluorescein labels a pair of proximal cysteines on the Ca2+-ATPase of sarcoplasmic reticulum.
Previous energy transfer studies [Squier, T. C., Bigelow, D. J., de Ancos, J. G., & Inesi, G. (1987) J. Biol. Chem. 262, 4748-4754] have utilized fluorescent iodoacetamide derivatives covalently bound to the Ca2+-ATPase of sarcoplasmic reticulum (SR), using labeling conditions that completely modify the most reactive of the protein's surface sulfhydryls to a final level of 9 nmol/mg of SR protein. Unambiguous interpretation of these results requires localization of these labeling sites with respect to the primary structure of the Ca2+-ATPase. In the present study, we have used the probe 6-(iodoacetamido)fluorescein (IAF) as a marker for these sites. The IAF-labeled Ca2+-ATPase was completely proteolyzed with trypsin, followed by centrifugation to remove (unlabeled) membrane-associated portions. The soluble IAF-labeled tryptic peptides were purified by size-exclusion and reverse-phase high-performance liquid chromatography. Two IAF-peptides resulted. The major (4.1 nmol of IAF/mg of starting protein) and minor (1.9 nmol/mg) IAF-peptides were sequenced and were identified, respectively, as Ala673-IAF-Cys674-Cys675-Phe676-Ala677+ ++-Arg678 and as Glu668-Ala669-IAF-Cys670-Arg671. A model is proposed to explain the selectivity of IAF for Cys670 and Cys674 of the approximately 14 surface sulfhydryls of the Ca2+-ATPase. The labeling region, Arg667 through Arg678, has been predicted to be alpha-helical; Cys670 and Cys674 would be adjacent in the helix and imbedded in an Arg cluster. The Arg residues would both attract the anionic IAF and enhance sulfhydryl reactivities by lowering their pK values.
Animals↗
Inhibition of sarcoplasmic reticulum Ca2+-ATPase by Mg2+ at high pH.
Steady state turnover of Ca2+-ATPase of sarcoplasmic reticulum has generally been reported to have a bell-shaped pH profile, with an optimum near pH 7.0. While a free [Mg2+] of 2 mM is optimal for activity at pH 7.0, it was found that this level was markedly inhibitory (K1/2 = 2 mM) at pH 8.0, thus accounting for the generally observed low activity at high pH. High activity was restored at pH 8.0 using an optimum free [Mg2+] of 0.2 mM. The mechanism of the Mg2+-dependent inhibition at pH 8.0 was probed. Inhibition was not due to Mg2+ competition with Ca2+ for cytoplasmic transport sites nor to inhibition of formation of steady state phosphoenzyme from ATP. Mg2+ inhibited (K1/2 = 1.8 mM) decay of steady state phosphoenzyme; thus, the locus of inhibition was one of the phosphoenzyme interconversion steps. Transient kinetic experiments showed that Mg2+ competitively inhibited (Ki = 0.7 mM) binding of Ca2+ to lumenal transport sites, blocking the ability of Ca2+ to reverse the catalytic cycle to form ADP-sensitive, from ADP-insensitive, phosphoenzyme. The data were consistent with a hypothesis in which Mg2+ binds lumenal Ca2+ transport sites with progressively higher affinity at higher pH to form a dead-end complex; its dissociation would then be rate-limiting during steady state turnover.
Adenosine Triphosphate↗