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The present study was designed to investigate the ameliorating effect of aqueous extract of C. dactylon on carbofuran induced oxidative stress (OS) and alterations in the activity of acetylcholinesterase (AChE) in the brain of rats. Vitamin C was used as a positive control. Wistar rats were administered with single sub-acute oral dose (1.6 mgkg-1 b.wt.) of carbofuran for 24 h. The OS parameters such as lipid peroxidation (LPO) and the activities of antioxidant enzymes including super oxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST), and that of AChE were studied in brain. Carbofuran treatment significantly increased the activities of SOD and CAT by 75 and 60%, respectively. It also induced the level of LPO by 113%. In contrast, the activities of GST and AChE were recorded to be diminished by 25 and 33%, respectively. Pretreatment of the rats with aqueous extract of C. dactylon (oral; 500mgkg-1) restored SOD activity completely but CAT activity only partially (7%). Carbofuran induced LPO was moderated by 95% in the brain of C. dactylon treated rats. The observed changes in OS parameters in C. dactylon treated group were comparable to that observed in vitamin C (200 mg-kg-1 b. wt.) treated group. Surprisingly, C. dactylon treatment significantly recovered the activity of AChE to a similar level as observed in the brain of control group. In contrast vitamin C treatment did not cause significant change in the activity of AChE in carbofuran treated group. There were no noticeable changes in the aforementioned study parameters in the brain of rats receiving C. dactylon and vitamin C, only. The results suggest that the study is extremely important in the context of development of new anticholinestesterase and antioxidant antidotes against carbofuran from C. dactylon.

The aim of this investigation was to prepare clonazepam microemulsions (CME) for rapid drug delivery to the brain to treat acute status epileptic patients and to characterize and evaluate the performance of CME in vitro and in vivo in rats. The CME were prepared by the titration method and were characterized for globule size and size distribution, zeta potential, and drug content. CME was radiolabeled with (99m)Tc (technetium) and biodistribution of drug in the brain was studied in Swiss albino rats after intranasal and intravenous administrations. Brain scintigraphy imaging in rabbits was also performed to ascertain the uptake of the drug into the brain. Pre and postCME formulation treated human nasal mucosa was subjected to transmission electron microscopy to investigate the mechanism of drug uptake across the nasal mucosa. CME were transparent and stable with mean globule size of 15 +/- 10 nm and zeta potential of -30 mV to -40 mV. (99m)Tc-labeled clonazepam solution ((99m)Tc CS)/ clonazepam microemulsion (CME)/clonazepam mucoadhesive microemulsion (CMME) were found to be stable and suitable for in vivo studies. Brain/blood uptake ratios at 0.50 hour (h) following intranasal CMME, CME, clonazepam solution (CS), and intravenous CME administrations were found to be 0.67, 0.50, 0.48, and 0.13, respectively indicating more effective targeting with intranasal administration and best targeting of the brain with intranasal CMME. Brain/blood ratio at all sampling points up to 8 h following intranasal administration of CMME compared to intravenous was found to be twofold higher indicating larger extent of distribution of the drug in brain. Rabbit brain scintigraphy also showed higher intranasal uptake of the drug into the brain. Transmission electron microscopy revealed significant accretion of CMME within interstitial spaces and paracellular mode of transport due to stretching of the tight junctions present in the nasal mucosa. This investigation demonstrates a more rapid and larger extent of transport of clonazepam into the rat brain with intranasal CMME, which may prove useful in treating acute status epileptics., (Copyright 2006 Wiley-Liss, Inc. and the American Pharmacists Association.)

The aim of this investigation was to prepare microemulsions containing sumatriptan (ST) and sumatriptan succinate (SS) to accomplish rapid delivery of drug to the brain in acute attacks of migraine and perform comparative in vivo evaluation in rats. Sumatriptan microemulsions (SME)/sumatriptan succinate microemulsions (SSME) were prepared using titration method and characterized for drug content, globule size and size distribution, and zeta potential. Biodistribution of SME, SSME, sumatriptan solution (SSS), and marketed product (SMP) in the brain and blood of Swiss albino rats following intranasal and intravenous (IV) administrations were examined using optimized technetium-labeled ((99m)Tc-labeled) ST formulations. The pharmacokinetic parameters, drug targeting efficiency (DTE), and direct drug transport (DTP) were derived. Gamma scintigraphy imaging of rat brain following IV and intranasal administrations were performed to ascertain the localization of drug. SME and SSME were transparent and stable with mean globule size 38 +/- 20 nm and zeta potential between -35 to -55 mV. Brain/blood uptake ratios at 0.5 hour following IV administration of SME and intranasal administrations of SME, SMME, and SSS were found to be 0.20, 0.50, 0.60, and 0.26, respectively, suggesting effective transport of drug following intranasal administration of microemulsions. Higher DTE and DTP for mucoadhesive microemulsions indicated more effective targeting following intranasal administration and best brain targeting of ST from mucoadhesive microemulsions. Rat brain scintigraphy endorsed higher uptake of ST into the brain. Studies conclusively demonstrated rapid and larger extent of transport of microemulsion of ST compared with microemulsion of SS, SMP, and SSS into the rat brain. Hence, intranasal delivery of ST microemulsion developed in this investigation can play a promising role in the treatment of acute attacks of migraine.

The aim of this investigation was to prepare microemulsions containing zolmitriptan (ZT) for rapid drug delivery to the brain to treat acute attacks of migraine and to characterize microemulsions and evaluate biodistribution in rats. Zolmitriptan microemulsions (ZME) were prepared using the titration method and were characterized for globule size distribution and zeta potential. ZT was radiolabeled using (99m)Tc (technetium) and radiolabeled-drug formulations of ZT were used to carry out biodistribution of drug in the brain of Swiss albino rats after intranasal and intravenous administration. The pharmacokinetic parameters, drug targeting efficiency (%DTE) and direct nose-to-brain drug transport (%DTP) were calculated. Brain scintigraphy imaging in rats were also performed to ascertain the uptake of drug into the brain. ZME were transparent and stable with mean globule size of 35 +/- 25 nm and zeta potential of - 38- - 52 mV. (99m)Tc-labeled-drug formulations of ZT were found to be stable and suitable to perform in vivo studies. Following intranasal administrations of zolmitriptan mucoadhesive microemulsion (ZMME), ZME, Zolmitriptan solution (ZS) and intravenous administration of ZS, brain/blood uptake ratios at 0.50 h were found to be 0.70, 0.56, 0.27 and 0.13, respectively, indicating effective brain-targeting following intranasal administration of ZMME. Comparing intranasal administration of ZMME with intravenous administration of ZME, the %DTE and %DTP were found higher indicating effective drug transport following intranasal administration and highest brain-targeting following ZMME administration. Rat brain scintigrams showed substantial uptake of drug into the brain after intranasal administration of ZMME. Studies of this investigation conclusively demonstrated rapid and larger extent of transport into the rat brain following intranasal administration of ZMME and can play a promising role in the treatment of acute attacks of migraine.

The amplification of cyclic nucleotide 'second messenger' signals within neurons is controlled by phosphodiesterases which are responsible for their degradation. Calmodulin-dependent phosphodiesterase (CaMPDE) is an abundant enzyme in brain which carries out this function. For the first time, we have localized CaMPDE in the normal human brain at various ages, using a mononoclonal antibody designated A6. This antibody was generated using standard techniques, purified, and applied to tissue sections. Autopsy specimens of human brain with no neuropathological abnormalities were selected representing a range of pre- and postnatal ages. Sections of various brain regions were evaluated for immunoreactivity, graded as nil, equivocal, or definite. We demonstrated definite CaMPDE immunohistochemical staining in neocortex, especially in neurons in layers 2 and 5. There was definite neuronal immunoreactivity in the hippocampus, and in the subiculum. The striatum had definite patchy neuronal staining. Definite terminal staining in the globus pallidus externa and substantia nigra pars reticulata outlined resident neurons, interpreted as axonal terminal staining. Cerebellar Purkinje cells showed definite immunoreactivity. In the developing brain, definite immunohistochemical staining was seen in the cerebellar external granular layer. The expression of CaMPDE in specific subsets of neurons suggests they may correlate with cells having dopaminergic innervation and/or high levels of neuronal integration.

In the present study we have shown that bovine brain 60-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme (CaMPDE - PDE1A2) is proteolyzed by a Ca2+-dependent cysteine protease, m-calpain. The proteolysis of PDE1A2 by m-calpain results in its conversion to a totally calmodulin (CaM)-independent form accompanied by degradation of PDE1A2 into a 45-kDa catalytic fragment and a 15-kDa fragment. The activity of PDE1A2 is unaffected by the presence or absence of CaM during cleavage, suggesting that the interaction between CaM and PDE1A2 does not alter substrate recognition by calpain. Furthermore, we provide evidence, based on the studies of CaM overlay and phosphorylation, that the cleavage site is not present either in the CaM-binding domain or phosphorylation site. N-terminal sequence analysis of the 45-kDa fragment indicated that cleavage occurs between residues 126Gln and 127Ala, and eliminates the CaM-dependent activity of carboxy termini PDE1A2. The present findings suggest that limited proteolysis in the brain through calpains could be an alternate mechanism for activating CaMPDE(s) and for regulating intracellular levels of cAMP., (Copyright 1998 Academic Press.)

The effect of amantadine (an antiparkinsonian agent) on calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes was investigated. Amantadine inhibited bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase but not the bovine brain 63 kDa, heart and lung calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes. The inhibition of bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase was overcome by increasing the concentration of calmodulin. This suggests that amantadine may be an antagonist of calmodulin or act specifically and reversibly on the action of calmodulin. The bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme is predominantly expressed in the brain and its inhibition may result in increased intracellular levels of cyclic AMP (cAMP). The increased intracellular levels of cAMP have a protective role for dopaminergic neurons. The present findings suggest that amantadine may be a valuable tool to investigate the physiological role of 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme in the progression of Parkinson's disease and gives a new insight into the action of this drug.

Intracellular concentrations of cyclic nucleotides is regulated by cyclic nucleotide phosphodiesterases and calmodulin-dependent cyclic nucleotide phosphodiesterases (CaMPDE), one of the most intensively studied and best characterized phosphodiesterases. In the present study, the effect of an antiparkinsonian agent, deprenyl (selegeline hydrochloride) which is believed to be a selective inhibitor of monoamine oxidase-B, on bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) isozymes have been investigated. The findings indicated that deprenyl inhibited brain 60 kDa isozyme, however the inhibition for brain 63 kDa CaMPDE was observed to a lesser extent. The inhibition of brain 60 kDa CaMPDE was overcome by increasing the concentration of calmodulin suggesting that deprenyl may be calmodulin antagonist or act specifically and reversibly on the action of calmodulin. The 60 kDa CaMPDE isozyme is predominantly expressed in brain and its inhibition can result in increased intracellular levels of cAMP. The increased intracellular levels of cAMP have a protective role for dopaminergic neurons. Therefore, deprenyl may be a valuable tool to investigate the physiological roles of brain CaMPDE isozymes in progression of Parkinson's disease and gives a new insight into the action of this drug.

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyses the transfer of myristate from myristoyl-CoA to the NH2-terminal glycine residue of a number of important proteins of diverse function. Little is known about the control and regulation of NMT in higher eukaryotes. We have identified a N-myristoyltransferase activator (NAF45) which copurified with one form of bovine brain NMT activity which failed to bind to phosphocellulose column chromatography (NMT.PU) and represented 32 +/- 4% of the total NMT activity applied to this column. The NAF45 and NMT activities were resolved by mono Q column chromatography from the NMT.PU fraction. Resolution resulted in a loss of NMT.PU activity. NAF45 was a nondialysable molecule with an apparent molecular mass of 45 kDa. Reconstitution of the NAF45 and NMT resulted in the recovery of NMT activity, indicating that this form of NMT is dependent on the presence of NAF45. NAF45 was able to stimulate other forms of NMT activity (maximum of 3-4 fold) in a highly cooperative fashion (Hills coefficient of approximately 5.5). Stimulation was in terms of both the rate of myristoylation and the overall production of myristoylpeptide, indicating that the end point of the reaction was shifted in favour of myristoylpeptide production. NAF45 was able to compete with NIP71 for NMT [King, M.J. and Sharma, R.K. (1993) Biochem, J. 291, 635-639], although its affinity appeared lower than the NIP71.

N-Myristoyltransferase is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the NH2-terminal glycine residue of a number of protein of diverse functions. Many of the known myristoylated proteins are important in signal transduction. We have compared the activity of rat liver N-myristoyltransferase from lean and obese (fa/fa) Zucker rats (a model for non-insulin dependent diabetes mellitus, NIDDM). N-myristoyltransferase activity isolated from the particulate fraction of obese (fa/fa) Zucker rat liver was approximately 4.7-fold lower than the corresponding activity observed in either the controls or the vanadate-treated obese rat livers. This pattern was only observed in the particulate fraction; the homogenate and soluble N-myristoyltransferase activities were not significantly different to the control activities. N-myristoyltransferase activity isolated from the brain of the four groups showed no significant variations. These results, and previous work [King, M. J., Pugazhenthi, S., Khandelwal, R. L. and Sharma, R. K. (1993) Biochim. Biophys. Acta. 1165, 259-262], would indicate that the rat liver particulate N-myristoyltransferase activity appears to be inversely proportional to the level of plasma insulin, implicating insulin in the control of N-myristoylation. The specific activity of the particulate liver N-myristoyltransferase was approximately 10-fold higher than that of the soluble liver N-myristoyltransferase, raising the possibility that N-myristoyltransferase exists in 2 populations, with the active form of N-myristoyltransferase residing in the membranous fraction. This situation could provide a system whereby N-myristoylation is regulated by the translocation of N-myristoyltransferase from the cytosol to its active site in the membranes.

Bovine brain contains two calmodulin-dependent phosphodiesterase kinases which are separated on Sephacryl S-300 column. One of these kinases has been purified to homogeneity and shown to belong to the calmodulin-dependent protein kinase II family. Phosphorylation of the 63 kDa phosphodiesterase by this purified protein kinase results in the incorporation of 1.0 mol phosphate per mol subunit and an accompanying increase in Ca2+ concentrations required for the phosphodiesterase activation by calmodulin. The protein kinase undergoes autophosphorylation to incorporate 1.0 mol phosphate per mol of subunit of the enzyme and the autophosphorylated enzyme is active, independent of the presence of Ca2+. The autophosphorylation reaction as well as the protein kinase reaction are rendered Ca2+ independent in less than 15 seconds when approximately one mol phosphate per mol protein kinase is incorporated. The result suggests that activation of phosphodiesterase phosphorylation reaction may occur prior to the activation of phosphodiesterase and phosphatase during a cell Ca2+ flux via the protein kinase autophosphorylation mechanism.

A bovine brain calmodulin-dependent protein kinase undergoes autophosphorylation with a maximal incorporation of 10 mol phosphate per mol enzyme. The autophosphorylated enzyme is fully active, independent of Ca2+ and calmodulin. Although the autophosphorylation reaction depends on Ca2+ and calmodulin, it can be rendered Ca(2+)-independent after about 15 seconds when 1 mol phosphate has been incorporated per mol enzyme. This Ca(2+)-independent autophosphorylation is an intramolecular reaction, since mixing of autophosphorylated and non-phosphorylated calmodulin-dependent protein kinase does not result in the Ca(2+)-independent phosphorylation of the non-phosphorylated enzyme. The Ca(2+)-independent autophosphorylation can be inhibited by an exogenous substrate, e.g., casein. The casein phosphorylation activity is approximately proportional to the level of phosphorylation of the enzyme.

Four distinct N-myristoyl transferase (NMT) activity peaks, designated I, II, III, and IV, were separated from the cytosolic fraction of bovine brain by DEAE-Sepharose column chromatography. Peaks I, II, III and IV were characterised biochemically with respect to substrate specificity: with cAMP-dependent protein kinase and pp60src derived peptides, and by their apparent molecular mass. The apparent molecular mass of peaks I, II, III and IV were 190 kDa, 224 kDa, 390 kDa and 76 kDa, respectively. These results indicate that bovine brain contains multiple forms of NMT.

N-myristoyl-CoA:protein N-myristoyl transferase is the enzyme that catalyzes the covalent transfer of myristic acid to the NH2-terminal glycine residue of a protein, or peptide, substrate. We have established a new, rapid, reliable, and inexpensive myristoyl-CoA:protein N-myristoyl transferase assay. This N-myristoyl transferase assay is based on the binding of the [3H]myristoylated peptide to a P81 phosphocellulose paper matrix and is more convenient for assaying multiple samples than existing procedures. Two peptides, derived from the N-terminal sequences of the type II catalytic subunit of cAMP-dependent protein kinase and pp60src, were used as substrates. A survey of rat and bovine tissue extracts demonstrated that in both cases brain contained the highest NMT activity (i.e., brain greater than spleen greater than heart greater than liver). Under the assay conditions used, the rate of myristoylation was linear for 10 min and with up to 4.0 mg/ml of brain extract.

Inhibition of a purified 60 KDa bovine brain calmodulin-dependent cGMP phosphodiesterase (PDE) was investigated for a number of peptides and non-peptides which are known to bind to angiotensin (ANG) receptors. The peptide antagonists sarilesin and sarmesin had KI = 120 and greater than 200 microM respectively, and the peptide agonists ANG II and ANG III had KI = greater than 200 and 45 microM respectively. Non-peptide ANG receptor antagonists related to DuP 753 exhibited KI values in the same range. For both peptide and non-peptide antagonists, inhibitory activities in the PDE assay reflected the order of antagonist potencies at ANG receptors in the rat isolated uterus assay and binding affinities at ANG receptors in rat uterine membranes, suggesting that molecular recognition factors are similar for both ANG receptors and cGMP PDE. The vasodilatory and blood pressure lowering effects of compounds related to DuP 753 may be due in part to inhibition of cGMP PDE. The differential effects of ANG II and ANG III at target tissues may relate in part to the marked differences in cGMP PDE inhibition associated with these two peptides hormones.

Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was purified more than 3,000-fold from the brain extracts with greater than 12% yield. The purified phosphodiesterase could be activated 10- to 15-fold by calmodulin and Ca2+ to a specific enzyme activity of more than 300 mumol of cAMP hydrolyzed/min/mg of protein. Molecular weight of the enzyme was determined to be 115,800 by the sedimentation equilibirum method or 124,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight of 58,000. These results suggested that the calmodulin-dependent phosphodiesterase from bovine brain has a subunit structure of alpha2. Molecular weight of the complex of calmodulin and phosphodiesterase was the complex of calmodulin and phosphodiesterase was also calculated from the sedimentation constant and Stokes radius to be 159,000. Since calmodulin has a molecular weight of about 17,000, the result indicated that the stoichiometry of the complex is calmodulin2 alpha2. The catalytic subunit of cylic AMP-dependent protein kinase was found to catalyze the phosphorylation of the purified phosphodiesterase with the incorporation of 2 mol of phosphate/mol of the enzyme.

Purified bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) contains isozymes that are composed of two distinct subunits with molecular masses of 60,000 and 63,000 daltons. Analysis by NaDodSO4 gel electrophoresis and autoradiography of a phosphodiesterase sample phosphorylated in the presence of [32P]ATP and bovine heart cAMP-dependent protein kinase catalytic subunit revealed that only the 60-kDa subunit was phosphorylated. By using an isozyme preparation greatly enriched with the 60-kDa subunit, the following observations regarding the subunit phosphorylation were made. First, the phosphorylation resulted in the maximal incorporation of about 2 mol of phosphate per mol of subunit. Second, complete inhibition of 60-kDa subunit phosphorylation was approached at a saturating concentration of Ca2+ when a molar ratio of calmodulin to phosphodiesterase of 2:1 was used. No inhibition was observed in the presence of either Ca2+ or calmodulin alone. Third, the phosphorylation was accompanied by a decrease in the enzyme affinity for calmodulin; calmodulin concentrations required for 50% activation of nonphosphorylated and maximally phosphorylated phosphodiesterase isozyme samples were 0.51 and 9.3 nM, respectively. Fourth, the phosphodiesterase isozyme could be dephosphorylated by the calmodulin-dependent phosphatase (calcineurin) in the presence of Ni2+ or Mn2+, the dephosphorylation being associated with an increase in the enzyme affinity for calmodulin. Fifth, peak II rabbit liver phosphoprotein phosphatase catalytic unit did not catalyze the dephosphorylation of the phosphodiesterase isozyme.

An inhibitor protein of cyclic nucleotide phosphodiesterase is demonstrated in bovine brain extract and separated from modulator binding protein, a recently discovered inhibitory factor of phosphodiesterase. The new inhibitor protein is similar to the cyclic AMP phosphodiesterase inhibitor from bovine retina (Dumler, I. L., and Etingof, F. N. 1976) Biochim. Biophys, Acta 429, 474-484) in its heat stability: it retains full activity upon heating in a boiling water bath for 2 min. The new inhibitor protein counteracts the activation of the Ca2+-activatable cyclic nucleotide phosphodiesterase by the Ca2+-dependent modulator protein without affecting the basal activity of the enzyme. The inhibition of phosphodiesterase by the inhibitor can be reversed by high concentrations of modulator protein but is not influenced by a 20-fold increase in Ca2+ concentration. In contrast, a Ca2+-independent form of cyclic nucleotide phosphodiesterase is not inhibited by the inhibitor protein. These results suggest that the heat-stable inhibitor protein is specific against the action of the Ca2+-dependent modulator protein. Gel filtration analyses on Sephadex G-75 and G-100 columns have shown that the inhibitor protein and the modulator protein may associate in the presence of Ca2+. The molecular weights determined by the gel filtration for the free inhibitor protein and the complex of the inhibitor and modulator protein are about 70,000 and 85,000, respectively.

Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, from the bovine heart and brain, purified by monoclonal antibody chromatography were tested with respect to activation by oncomodulin. The heart and brain enzymes which have previously been shown to have slightly different electrophoretic mobilities (1), were found to also differ in the oncomodulin dose-dependent activation of cAMP hydrolysis. Oncomodulin was shown to activate the heart enzyme to the same extent as calmodulin. However, this study indicates that the heart phosphodiesterase has approximately 25-fold higher affinity for oncomodulin than the brain enzyme. The oncomodulin concentration required for the half-maximal activation of the heart phosphodiesterase was estimated to be 2 X 10(-7)M. In addition, the possibility of the observed activation by oncomodulin being due to calmodulin contamination can be ruled out as the oncomodulin activation profiles were unaltered subsequent to chromatography on organomercurial agarose and the activation by oncomodulin could not be reversed by anti-calmodulin IgG.

Bovine brain contains two major calmodulin (CaM) dependent phosphodiesterase isozymes which are homodimeric proteins with subunit molecular masses of 60 and 63 kilodaltons (kDa), respectively. The 60-kDa subunit isozyme can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme affinity towards CaM. The phosphorylation is blocked by Ca2+ and CaM and reversed by the CaM-stimulated phosphatase (calcineurin). The 63-kDa subunit isozymes can also be phosphorylated, but in this case by a CaM-dependent protein kinase(s). This phosphorylation is also accompanied by a decrease in the isozyme affinity towards CaM and can be reversed by the CaM-dependent phosphatase. Analysis of the complex regulatory properties of the phosphodiesterase isozymes has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaM-dependent phosphodiesterase isozymes play key roles in this signal coupling phenomenon.

Calmodulin-dependent cyclic nucleotide phosphodiesterase from bovine brain is found to be composed of two distinct subunits, 60,000- and 63,000-dalton polypeptides. Peptide mapping of the subunits by partial proteolysis demonstrated that the 60-kDa polypeptide is not derived from the 63-kDa species. The interaction of the enzyme with three monoclonal antibodies, A6, C1, and A2, and the analysis of immunocomplexes by sucrose density gradient centrifugation revealed that calmodulin-dependent cyclic nucleotide phosphodiesterase exists in three different forms, i.e. (a) homodiamer of 60-kDa, (b) heterodimer of 60- and 63-kDa, and (c) homodimer of 63-kDa. A6 antibody reacts with both 60- and 63-kDa polypeptides indicating that they are immunologically related. C1 and A2 antibodies react with only 60-kDa polypeptide species. By using C1 Sepharose 4B affinity column chromatography, the 63-kDa homodimer which did not bind to the column (Fraction I) was separated from the 60-kDa polypeptide containing isozymes (the heterodimer and the 60-kDa homodimer) which were retained on the column and later eluted as a mixture (Fraction II). Fraction I, the 63-kDa homodimer enzyme, has higher Vmax toward cGMP as substrate than cAMP whereas the opposite was found with Fraction II. The specific activity of Fraction II enzyme toward cAMP was approximately 500 mumol/min/mg, the highest value ever reported for brain calmodulin-dependent cyclic nucleotide phosphodiesterase preparations.

Bovine brain contains calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes which are composed of two distinct subunits: Mr 60,000 and 63,000. The 60-kDa but not the 63-kDa subunit-containing isozyme can be phosphorylated by cAMP-dependent protein kinase resulting in decreased affinity of this subunit toward calmodulin (Sharma, R. K., and Wang, J. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2603-2607). In contrast, purified 63-kDa subunit-containing isozyme has been found to be phosphorylated by a preparation of bovine brain calmodulin-binding proteins in the presence of Ca2+ and calmodulin. The phosphorylation resulted in the maximal incorporation of 2 mol of phosphate/mol of the phosphodiesterase subunit with a 50% decrease in the enzyme affinity toward calmodulin. At a constant calmodulin concentration of 6 nM, the phosphorylated isozyme required a higher concentration of Ca2+ for activation than the nonphosphorylated phosphodiesterase. The Ca2+ concentrations at 50% activation by calmodulin of the nonphosphorylated and phosphorylated isozymes were 1.1 and 1.9 microM, respectively. Phosphorylation can be reversed by the calmodulin-dependent phosphatase, calcineurin, but not by phosphoprotein phosphatase 1. The results suggest that the Ca2+ sensitivities of brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes can be modulated by protein phosphorylation and dephosphorylation mechanisms in response to different second messengers.