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M K Thomas

Publications and source records attributed to M K Thomas.

At least 19 recordsLinked to original sources
PubMed2006

Effect of Ocimum sanctum Linn. on cardiac changes in rats subjected to chronic restraint stress.

Male Wistar rats were subjected to chronic restraint stress (CRS; 6 h/day for 21 days) alone or along with either hydroalcoholic extract of Ocimum sanctum (Os; 100 mg/kg; orally) or MK-801, an NMDA receptor antagonist (0.3 mg/kg; i.p.). In the rats subjected to only CRS, plasma cAMP level was significantly raised on day 21, with no significant change in plasma corticosterone level. There was a significant (p < 0.05) fall in myocardial glutathione level, along with a significant (p < 0.05) rise in myocardial superoxide dismutase (SOD) and catalase activities, while light microscopy showed evidence of myocardial edema. Both Os and MK-801 significantly prevented the CRS-induced rise in plasma cAMP level, myocardial SOD and catalase activities as well as the light microscopic changes in the myocardium. This study revealed that Ocimum sanctum protects rat heart from chronic restraint stress induced changes, through its central effect.

Administration, Oral↗
PubMed2006

Correlation between inflammation and oxidative stress in normocholesterolemic coronary artery disease patients 'on' and 'off' atorvastatin for short time intervals.

AIM OF THE STUDY: To assess whether variations in antioxidant and anti-inflammatory parameters occur with short term administration and discontinuation of atorvastatin in normocholesterolemic coronary artery disease (CAD) patients. METHODS: Forty CAD patients with near normal serum cholesterol levels (total cholesterol <240 mg/dl, LDL cholesterol <130 mg/dl) were continuously enrolled and randomized to groups A and B (20 patients taking atorvastatin) and groups C and D (20 patients not taking atorvastatin). Atorvastatin (10 mg/day) was continued in group A, withdrawn in group B and started in groups C and D for 6 weeks. Thereafter atorvastatin was withdrawn in group A and C, restarted in group B, and continued in group D for further 6 weeks. CRP, FRAP and TBARS were assessed at baseline, 6 weeks and 12 weeks in all the groups. RESULTS: Baseline CRP, TBARS and FRAP levels were significantly different (p < 0.05) between groups A and B and C and D at the time of enrollment, indicating lower levels of oxidative stress (FRAP-172.40 +/- 23.41 nmol Fe(2+)/l vs 142.62 +/- 15.73 nmol Fe(2+)/l and TBARS-3.66 +/- 1.14 nmol/ml vs 6.11 +/- 1.85 nmol/ml) and low grade inflammation (CRP-1.38 +/- 0.69 mg/l vs 3.19 +/- 1.77 mg/l) in statin treated groups. In group B, discontinuation resulted in increase in CRP (2.87 +/- 0.98 mg/l) and TBARS (5.75 +/- 1.35 nmol/ml) and decrease in FRAP (133.132 +/- 13.32 nmol Fe(2+)/l) and whereas group A patients did not show significant variation in values compared to baseline (CRP-1.36 +/- 0.33 mg/l, FRAP-155.45 +/- 19.51 and TBARS-4.22 +/- 0.81). Administration of atorvastatin caused a marked reduction in oxidative stress and inflammation in groups C and D (CRP-1.13 +/- 0.99 mg/l and 1.73 +/- 1.60 mg/l, FRAP-166.54 +/- 34.11 and 177.44 +/- 13.31 nmol Fe(2+)/l, TBARS-4.66 +/- 2.33 and 3.55 +/- 1.25 nmol/ml respectively). The values returned to pretreatment levels on discontinuation of the drug in group C (CRP-2.61 +/- 1.28 mg/l, FRAP-138.49 +/- 19.62 nmol Fe(2+)/l, TBARS-6.13 +/- 0.74 nmol/ml) whereas the decline was maintained in group D (CRP-1.62 +/- 1.48 mg/l, FRAP-173.07 +/- 9.03 nmol Fe(2+)/l, TBARS-3.75 +/- 1.03 nmol/ml). CONCLUSION: Administration and withdrawal of atorvastatin caused changes in markers of oxidative stress which closely correlated with changes in marker of inflammation. Further, the salutary effects were of quick onset, but were rapidly reversed on withdrawal of atorvastatin.

Atorvastatin↗
PubMed2001

Development of diabetes mellitus in aging transgenic mice following suppression of pancreatic homeoprotein IDX-1.

Monogenic forms of diabetes can result from mutations in genes encoding transcription factors. Mutations in the homeodomain transcription factor IDX-1, a critical regulator of pancreas development and insulin gene transcription, confer a strong predisposition to the development of diabetes mellitus in humans. To investigate the role of IDX-1 expression in the pathogenesis of diabetes, we developed a model for the inducible impairment of IDX-1 expression in pancreatic beta cells in vivo by engineering an antisense ribozyme specific for mouse IDX-1 mRNA under control of the reverse tetracycline transactivator (rtTA). Doxycycline-induced impairment of IDX-1 expression reduced activation of the Insulin promoter but activated the Idx-1 promoter, suggesting that pancreatic beta cells regulate IDX-1 transcription to maintain IDX-1 levels within a narrow range. In transgenic mice that express both rtTA and the antisense ribozyme construct, impaired IDX-1 expression elevated glycated hemoglobin levels, diminished glucose tolerance, and decreased insulin/glucose ratios. Metabolic phenotypes induced by IDX-1 deficiency were observed predominantly in male mice over 18 months of age, suggesting that cellular mechanisms to protect IDX-1 levels in pancreatic beta cells decline with aging. We propose that even in the absence of Idx-1 gene mutations, pathophysiological processes that decrease IDX-1 levels are likely to impair glucose tolerance. Therapeutic strategies to attain normal glucose homeostasis by restoring normal IDX-1 levels may be of particular importance for older individuals with diabetes mellitus.

Aging↗
PubMed2001

Hedgehog signaling regulation of homeodomain protein islet duodenum homeobox-1 expression in pancreatic beta-cells.

Insulin gene expression in pancreatic beta-cells is regulated by signals from developmental morphogen proteins known as hedgehogs (Hhs). By analyzing 5'-deletion insulin promoter-reporter constructs in transient transfections of clonal INS-1 beta-cells, we located activating Hh-responsive regions within the rat insulin I promoter that include the glucose-response elements Far (E2) and Flat (A2/A3). Activation of Hh signaling in INS-1 cells by ectopic Hh expression increased (and inhibition of Hh signaling with the Hh-specific inhibitor cyclopamine decreased) transcriptional activation of a multimerized FarFlat enhancer-reporter construct. In DNA-binding studies, nuclear extracts from INS-1 cells activated by ectopic Hh expression increased (and extracts from INS-1 cells treated with cyclopamine decreased) protein binding to a radiolabeled FarFlat oligonucleotide probe. An antiserum directed against the transcription factor islet duodenum homeobox-1 (IDX-1), a regulator of pancreas development and activator of the insulin gene promoter, attenuated the binding activity of Hh-responsive protein complexes. Nuclear IDX-1 protein levels on Western blots were increased by ectopic Hh expression, thereby providing a mechanism for Hh-mediated regulation of the insulin promoter. Addition of cyclopamine to INS-1 cells decreased IDX-1 messenger RNA expression. In transient transfections of a -4.5-kb mouse IDX-1 promoter-reporter construct, ectopic Hh expression increased (and cyclopamine administration decreased) transcriptional activation of the IDX-1 promoter in a dose-dependent manner. Thus, the IDX-1 gene is a direct regulatory target of Hh signaling in insulin-producing pancreatic beta-cells. We propose that Hh signaling activates the insulin gene promoter indirectly via the direct activation of IDX-1 expression. Because IDX-1 gene expression is essential for insulin gene expression, pancreatic beta-cell development, and normal glucose homeostasis, our findings that Hh signaling regulates IDX-1 expression in the endocrine pancreas suggest possible novel therapeutic approaches for diabetes mellitus.

Animals↗
PubMed2001

Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes.

The endocrine cells of the rat pancreatic islets of Langerhans, including insulin-producing beta-cells, turn over every 40-50 days by processes of apoptosis and the proliferation and differentiation of new islet cells (neogenesis) from progenitor epithelial cells located in the pancreatic ducts. However, the administration to rats of islet trophic factors such as glucose or glucagon-like peptide 1 for 48 h results in a doubling of islet cell mass, suggesting that islet progenitor cells may reside within the islets themselves. Here we show that rat and human pancreatic islets contain a heretofore unrecognized distinct population of cells that express the neural stem cell-specific marker nestin. Nestin-positive cells within pancreatic islets express neither the hormones insulin, glucagon, somatostatin, or pancreatic polypeptide nor the markers of vascular endothelium or neurons, such as collagen IV and galanin. Focal regions of nestin-positive cells are also identified in large, small, and centrolobular ducts of the rat pancreas. Nestin-positive cells in the islets and in pancreatic ducts are distinct from ductal epithelium because they do not express the ductal marker cytokeratin 19 (CK19). After their isolation, these nestin-positive cells have an unusually extended proliferative capacity when cultured in vitro (approximately 8 months), can be cloned repeatedly, and appear to be multipotential. Upon confluence, they are able to differentiate into cells that express liver and exocrine pancreas markers, such as alpha-fetoprotein and pancreatic amylase, and display a ductal/endocrine phenotype with expression of CK19, neural-specific cell adhesion molecule, insulin, glucagon, and the pancreas/duodenum specific homeodomain transcription factor, IDX-1. We propose that these nestin-positive islet-derived progenitor (NIP) cells are a distinct population of cells that reside within pancreatic islets and may participate in the neogenesis of islet endocrine cells. The NIP cells that also reside in the pancreatic ducts may be contributors to the established location of islet progenitor cells. The identification of NIP cells within the pancreatic islets themselves suggest possibilities for treatment of diabetes, whereby NIP cells isolated from pancreas biopsies could be expanded ex vivo and transplanted into the donor/recipient.

Animals↗
PubMed2000

Vitamin D deficiency and disorders of vitamin D metabolism.

The disorders of vitamin D metabolism are inherited metabolic abnormalities involving mutations of the vitamin D receptor or enzymes involved in the metabolism of vitamin D to its biologically active form 1,25-dihydroxyvitamin D. Although these mutations are rare, studies in affected patients and animal models have helped to identify critical actions of vitamin D and the mechanism by which it exerts its effects. Vitamin D deficiency, however, is an increasingly recognized problem among the elderly and in the general population. Screening for vitamin D deficiency only in those patients with known risk factors will result in a large proportion of unrecognized affected patients.

Cholestanetriol 26-Monooxygenase↗
PubMed2000

Hedgehog signaling regulation of insulin production by pancreatic beta-cells.

Hedgehogs (Hhs) are intercellular signaling molecules that regulate tissue patterning in mammalian development. Mammalian Hhs include Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). The absence of Shh expression is required for the early development of the endocrine and exocrine pancreas, but whether Hh signaling functions in the fully developed adult endocrine pancreas is unknown. Here we report that Hhs Ihh and Dhh and their receptors patched (Ptc) and smoothened are expressed in the endocrine islets of Langerhans of the fully developed rat pancreas and in the clonal gamma-cell line INS-1. We demonstrate the coexpression of Ptc with insulin in beta-cells of mouse pancreatic islets, indicating that beta-cells are targets of active Hh signaling. The administration of cyclopamine, a Hh signaling inhibitor, decreases both insulin secretion from and insulin content of INS-1 cells. The effects of Hh signaling on insulin production occur at the transcriptional level because activation of Hh signal transduction by ectopic expression of Shh increases rat insulin I promoter activation in a dose-dependent manner in transient transfections of INS-1 and MIN6 beta-cell lines. In contrast, inhibition of Hh signaling with increasing concentrations of cyclopamine progressively reduces insulin promoter activity. Furthermore, the treatment of INS-1 cells with cyclopamine diminishes endogenous insulin mRNA expression. We propose that Hh signaling is not restricted to patterning in early pancreas development but also continues to signal in differentiated beta-cells of the endocrine pancreas in regulating insulin production. Thus, defective Hh signaling in the pancreas should be considered as a potential factor in the pathogenesis of type 2 diabetes.

Animals↗
PubMed1999

Bridge-1, a novel PDZ-domain coactivator of E2A-mediated regulation of insulin gene transcription.

Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neurogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancreatic beta cell, E2A proteins, in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for beta-cell function. By yeast two-hybrid screening of a cDNA library prepared from rat insulinoma (INS-1) cells, we identified a novel protein, Bridge-1, that interacts with E2A proteins and functions as a coactivator of gene transcription mediated by E12 and E47. Bridge-1 contains a PDZ-like domain, a domain known to be involved in protein-protein interactions. Bridge-1 is highly expressed in pancreatic islets and islet cell lines and the expression pattern is primarily nuclear. The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro-translated Bridge-1 with E12 or E47 and by mammalian two-hybrid studies. The PDZ-like domain of Bridge-1 is required for interaction with the carboxy terminus of E12. In both yeast and mammalian two-hybrid interaction studies, Bridge-1 mutants lacking an intact PDZ-like domain interact poorly with E12. An E12 mutant (E12DeltaC) lacking the carboxy-terminal nine amino acids shows impaired interaction with Bridge-1. Bridge-1 has direct transactivational activity, since a Gal4 DNA-binding domain-Bridge-1 fusion protein transactivates a Gal4CAT reporter. Bridge-1 also functions as a coactivator by enhancing E12- or E47-mediated activation of a rat insulin I gene minienhancer promoter-reporter construct in transient-transfection experiments. Substitution of the mutant E12DeltaC for E12 reduces the coactivation of the rat insulin I minienhancer by Bridge-1. Inactivation of endogenous Bridge-1 in insulinoma (INS-1) cells by expression of a Bridge-1 antisense RNA diminishes rat insulin I promoter activity. Bridge-1, by utilizing its PDZ-like domain to interact with E12, may provide a new mechanism for the coactivation and regulation of transcription of the insulin gene.

Amino Acid Sequence↗
PubMed1998

Hypovitaminosis D in medical inpatients.

BACKGROUND: Vitamin D deficiency is a major risk factor for bone loss and fracture. Although hypovitaminosis D has been detected frequently in elderly and housebound people, the prevalence of vitamin D deficiency among patients hospitalized on a general medical service is unknown. METHODS: We assessed vitamin D intake, ultraviolet-light exposure, and risk factors for hypovitaminosis D and measured serum 25-hydroxyvitamin D, parathyroid hormone, and ionized calcium in 290 consecutive patients on a general medical ward. RESULTS: A total of 164 patients (57 percent) were considered vitamin D-deficient (serum concentration of 25-hydroxyvitamin D, < or = 15 ng per milliliter), of whom 65 (22 percent) were considered severely vitamin D-deficient (serum concentration of 25-hydroxyvitamin D, <8 ng per milliliter). Serum 25-hydroxyvitamin D concentrations were related inversely to parathyroid hormone concentrations. Lower vitamin D intake, less exposure to ultraviolet light, anticonvulsant-drug therapy, renal dialysis, nephrotic syndrome, hypertension, diabetes mellitus, winter season, higher serum concentrations of parathyroid hormone and alkaline phosphatase, and lower serum concentrations of ionized calcium and albumin were significant univariate predictors of hypovitaminosis D. Sixty-nine percent of the patients who consumed less than the recommended daily allowance of vitamin D and 43 percent of the patients with vitamin D intakes above the recommended daily allowance were vitamin D-deficient. Inadequate vitamin D intake, winter season, and housebound status were independent predictors of hypovitaminosis D in a multivariate model. In a subgroup of 77 patients less than 65 years of age without known risk factors for hypovitaminosis D, the prevalence of vitamin D deficiency was 42 percent. CONCLUSIONS: Hypovitaminosis D is common in general medical inpatients, including those with vitamin D intakes exceeding the recommended daily allowance and those without apparent risk factors for vitamin D deficiency.

Adolescent↗
PubMed1998

Ligand-induced conformational changes in cyclic nucleotide phosphodiesterases and cyclic nucleotide-dependent protein kinases.

Three methods have been used to assess the conformational effects associated with ligand binding to two unrelated cyclic nucleotide receptor proteins: the cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) and the cGMP-dependent protein kinase (PKG). The methods should be applicable to other proteins and to other types of modification such as phosphorylation. The procedures use either ion-exchange chromatography, size-exclusion chromatography, or native gel electrophoresis of these proteins in the absence and presence of regulatory ligands. Measurements from these respective approaches allow documentation of changes in the quaternary structure, surface electronegativity, and relative compactness (Stokes radius) of the protein molecule. The combined data allow the changes in protein conformation to be quantitated in terms of alterations in the axial ratio or length/width dimension of the molecule. The methods can be applied to partially purified proteins and to proteins that are available in limited quantities. Conformational changes due to stable modifications of proteins can be potentially examined in crude extracts of intact cells. Each of the methods can be tailored to optimize resolution of a particular protein under a variety of conditions. Activity measurements, Coomassie brilliant blue or silver staining of gels, radioautography, or Western blot analysis can be used for detection of the protein.

1-Methyl-3-isobutylxanthine↗
PubMed1993

The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone.

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Systemic hypertension in infants with severe bronchopulmonary dysplasia: associated clinical factors.

The clinical course of 87 infants with bronchopulmonary dysplasia (BPD) on home oxygen therapy was reviewed to determine the occurrence of systemic hypertension (HTN) and to evaluate associated clinical features. Eleven of 87 (13%) infants developed systemic HTN either in the neonatal intensive care unit or following discharge. Clinical features that distinguished the hypertensive from the normotensive group were as follows: greater use of bronchodilators, 91% vs 37% (p < 0.001), and diuretics, 91% vs 55% (p < 0.05), longer duration of home oxygen therapy 21.6 +/- 9.9 vs 9.2 +/- 5.8 months (p < 0.05), and greater mortality, 36% vs 1% (p < 0.001). The course of systemic HTN in the surviving patients (7 of 11) was benign and resolved in all patients prior to weaning from home oxygen therapy. Systemic HTN is frequently present in infants with severe BPD and appears to be related to the clinical severity of lung disease.

Bronchopulmonary Dysplasia↗
PubMed1992

A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases.

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.

Amino Acid Sequence↗
PubMed1992

Kidney function in very low birth weight infants with furosemide-related renal calcifications at ages 1 to 2 years.

To determine whether long-term renal sequelae follow the use of furosemide in preterm infants, we evaluated renal function in 27 former very low birth weight infants (less than 1500 gm) at 1 to 2 years of age. Patients were classified into three groups on the basis of status at the time of discharge from the hospital: group 1 (n = 7) had no furosemide treatment or renal calcifications, group 2 (n = 10) had furosemide therapy but no calcifications, and group 3 (n = 10) had furosemide therapy with renal calcifications. Renal ultrasonography at the time of the study demonstrated resolution of the calcifications in six patients in group 3. No differences in renal function were observed between groups 1 and 2. Creatinine clearance (mean +/- SEM) in group 3 (83.6 +/- 7.8 ml/min per 1.73 m2) was significantly lower than clearance in groups 1 and 2 (103.2 +/- 6.5 and 109.1 +/- 5.1, respectively; p less than 0.05). Children in group 3 had significantly higher urinary calcium/creatinine ratios and fractional excretion of sodium and lower tubular reabsorption of phosphate than children in the two other groups had. Urine-blood difference in carbon dioxide tension after oral acetazolamide load, which indicates the ability of the distal tubule to secrete hydrogen ions, was 8.4 +/- 3.4 mm Hg in group 3, significantly lower than values in groups 1 and 2 (22.6 +/- 3.1 and 28.0 +/- 4.3 mm Hg, respectively, p less than 0.05). Within group 3 the four children with persistent renal calcifications had significantly lower urine-blood carbon dioxide tension differences than did those with resolution of calcifications (p = 0.02). We conclude that furosemide-related renal calcifications in very low birth weight infants may lead to glomerular and tubular dysfunction; further long-term follow-up of this population is recommended.

Absorption↗
PubMed1991

[Acceptance of headgear in children and teenagers].

This survey may provide a starting point for the improvement of the acceptability of headgears for children and teenagers during orthodontic treatment by taking aesthetic and material factors into consideration when selecting these appliances. The extent to which these findings are allowed to influence the planning of future treatment must be decided on an individual base; it is to be expected that such considerations will contribute both to the success of therapeutic aims and a reduction of costs in orthodontics.

Adolescent↗
PubMed1990

Characterization of a purified bovine lung cGMP-binding cGMP phosphodiesterase.

A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation.

1-Methyl-3-isobutylxanthine↗