Identification and purification of a 10-kilodalton protein associated with mitochondrial benzodiazepine receptors.

The isoquinoline carboxamide photoaffinity probe PK14105, a ligand with selectivity for mitochondrial benzodiazepine receptors, has been established to photolabel an 18-kDa protein. When this radioactive probe is used to photolabel rat mitochondrial preparations, a protein of 10 kDa, in addition to the 18-kDa protein, is identified following electrophoretic separation and extended autoradiography. These proteins are referred to herein as pk10 and pk18, respectively. Both proteins exhibited the same specificity to a series of ligands used in competition photolabeling studies and are mutually present at apparently similar ratios across multiple tissues. Subcellular fractionation of rat adrenals indicated that pk10 and pk18 comigrated with the mitochondrial marker enzyme cytochrome c oxidase. In numerous paradigms examining specificity, photolabeling of pk18 invariably coincided with photolabeling of pk10. In detergent-solubilized extracts of rat adrenal mitochondria, pk18 and pk10 coimmunoprecipitated when using antisera raised against pk18. Furthermore, purification of the photolabeled proteins using nondenaturing conditions demonstrated that pk18 and pk10 copurify substantiating their intimate association. A set of three antisera, specific to different regions of pk18, did not recognize pk10 on Western blots. Likewise, partial amino acid sequence of peptide fragments indicate that pk10 is not derived from proteolytic cleavage of pk18. These data suggest that pk10 represents another component of mitochondrial benzodiazepine receptors whose identity is not apparent with any known protein.

Studies on the phosphorylation of the 18 kDa mitochondrial benzodiazepine receptor protein.

Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein. In digitonin-permeabilized MA-10 Leydig tumor cells incubated with [gamma-32P]ATP, phosphorylation of MBR was detectable during treatment of the cells with dibutyryl cAMP. In conclusion, these data show that the MBR protein is an in vitro and in situ substrate of Protein kinase A, but the role of this phosphorylation in the regulation of steroidogenesis remains to be established.

Phosphodiesterase II, the cGMP-activatable cyclic nucleotide phosphodiesterase, regulates cyclic AMP metabolism in PC12 cells.

Analysis of cyclic nucleotide phosphodiesterase (PDE) activity in cellular fractions from cultured rat pheochromocytoma (PC12) cells has shown that the predominant hydrolytic activity in both cytosolic and particulate compartments is characteristic of a PDE II, the cGMP-activatable family of PDE isozymes. Cytosolic PDE activity was purified to a high degree utilizing DE-52 anion exchange and cGMP-Sepharose affinity chromatographies. The physicochemical properties of PC12 PDE II were similar to those of PDE II isolated from particulate or soluble fractions of other tissues, including subunit molecular weight of approximately 102,000, activation of cAMP hydrolysis by cGMP, and positive cooperative kinetic behavior for cAMP and cGMP hydrolysis. The potential role of PDE II in regulating cAMP metabolism in intact PC12 cells was studied using an [3H]adenine prelabeling technique. Stimulation of PC12 cell adenosine receptors resulted in a 5-8-fold increase in cAMP accumulation. Removal of the adenosine stimulus by the addition of exogenous adenosine deaminase resulted in a rapid decay of cAMP to prestimulated basal levels within 2 min. Treatment of PC12 cells with atrial natriuretic factor or sodium nitroprusside caused 1) increased intracellular cGMP levels, 2) attenuation of adenosine-stimulated cAMP accumulation, and 3) increased rates of cAMP decay after removal of the adenosine stimulus. Treatment of PC12 cells with HL-725 (a potent inhibitor of isolated PDE II activity in vitro) caused 1) increased basal cAMP accumulation, 2) potentiation of adenosine-stimulated cAMP accumulation, and 3) retardation of the rate of cAMP decay after removal of the adenosine stimulus. HL-725 blocked both the attenuation of cAMP accumulation and the accelerated rate of cAMP decay observed with the cGMP-elevating agents. These results suggest that, in PC12 cells, drugs or hormones that inhibit PDE II or increase intracellular cGMP levels to activate PDE II can modulate cAMP metabolism by altering the catalytic status of the enzyme.

Molecular properties of cyclic nucleotide phosphodiesterase isozymes.

Mammalian cells contain multiple molecular forms of cyclic nucleotide phosphodiesterase that differ in substrate specificity and kinetic and regulatory properties. Calcium/calmodulin and cyclic GMP are important regulators of the hydrolysis of cyclic AMP by either stimulating or inhibiting the activity of distinct forms of phosphodiesterase. Several isozymes of cyclic nucleotide phosphodiesterase have been purified to apparent homogeneity. Although some sequence homology is observed the isozymes appear genetically distinct by immunological criteria. Cyclic AMP- and calmodulin-dependent protein kinases can phosphorylate these enzymes and alter their kinetic and regulatory properties. Both tissue specificity and pharmacological selectivity of isozymes have been demonstrated for several drugs. In certain cases, e.g. cardiac muscle, the selective inhibition of a high affinity cAMP phosphodiesterase activity in a specific subcellular fraction correlates with pharmacologic responses. The results from molecular and pharmacologic studies of cyclic nucleotide phosphodiesterases have indeed expanded the role this system of isoenzymes exerts in the regulation of cellular function.

Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain.

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.

Correlation of cell-free brain cyclic nucleotide phosphodiesterase activities to cyclic AMP decay in intact brain slices.

Differential and gradient centrifugation of rat brain cerebral cortical homogenates show three cyclic nucleotide phosphodiesterase (CN PDE) activities localized to different subcellular fractions with varying relative specific activities and responsiveness to pharmacologic agents. Type I (calcium/calmodulin-activatable) CN PDE is found primarily in the cytosolic fraction, Type II (cGMP-activatable) CN PDE is predominately membrane associated, and Type IV (cGMP-insensitive) cAMP PDE is distributed equally between soluble and particulate fractions. Fractionation of cerebral cortical membranes shows that Type II and Type IV CN PDE activities reside in synaptosomes. Type II CN PDE is the predominate hydrolytic activity in synaptosomes whereas Type IV cAMP PDE contributes only a small percentage of the total cAMP hydrolysis and Type I CN PDE is not detected in this fraction. The contribution of CN PDE isozymes to the regulation of intracellular cAMP levels was studied using rat brain cortical slices. The rate of cAMP decay in the absence and presence of selective CN PDE inhibitors after adenosine or beta-adrenergic agonist stimulation was determined using an adenine prelabeling technique. These studies show that a rolipram-sensitive, high affinity cAMP PDE (Type IV) is principally responsible for cyclic AMP decay in intact cortical tissue following elevation of cyclic AMP levels by either adenosine or beta-adrenergic receptor agonists. However, this isozyme, which is sensitive to inhibition by rolipram, RO 20-1724 and SQ 65442 contributes only a small percentage of the total cAMP hydrolytic activity in cell-free preparations of cortex.