Localization of 3′,5′-cyclic adenosine monophosphate phosphodiesterase (cAMP-PDEase) activity in isolated bovine thyroid plasma membranes

Summary Isolated bovine thyroid plasma membrane preparations were obtained by isopycnic density gradient centrifugution. Cyclic AMP-PDEase (EC 3.1.4.c) activity has been demonstrated by electron microscopic histochemistry on the unit membrane of isolated bovine thyroid cells. 3-isobutyl-1-methyl-xanthine (IBMX) produced partial inhibiton, while omission of the substrate revealed no reaction product deposition. These observations correlated well with biochemical studies that showed 0.4% of the total cAMP-PDEase activity to be present in the plasma membrane preparations. Kinetic analysis of cAMP hydrolysis yielded two apparent Michaelis constants for the homogenate and the plasma membrane-rich fraction. Dose-response curves for IBMX inhibition showed cAMP-PDEase of the homogenate to be more sensitive to inhibition than that of the plasma membrane-bound enzyme. Furthermore, wash experiments indicate that the plasma membrane-associated enzyme is tightly bound. This investigation strengthens our previous study and suggests that bovine thyroid cell plasma membranes contain a cAMP-PDEase that may be involved in interactions between the cell and the external environment in a manner yet to be determined.     

Relations between plasma membrane and lysosomal membrane. 2. Quantitative evaluation of plasma membrane marker enzymes in the lysosomes

We have quantified, in cultured rat fibroblasts, the association to the lysosomal membrane of two classical plasma membrane markers, 5'-nucleotidase and alkaline phosphodiesterase I. To isolate highly purified lysosomal preparations, lysosomes were loaded with horseradish peroxidase (2-h cell uptake, 16-h chase) and isolated by isopycnic centrifugation in linear Percoll gradients, followed by a 3,3'-diaminobenzidine-induced density shift in sucrose gradients. Purified lysosomal preparations contained up to 50% of N-acetyl-beta-glucosaminidase of the homogenate. This lysosomal enzyme was enriched 33-fold in the most purified preparations. In the electron microscope, these preparations appeared to be highly purified and only contained organelles filled with diaminobenzidine reaction products. Analysis of purified preparations indicates that 0.5-0.8% of 5'-nucleotidase, but as much as 10.9-14.3% of alkaline phosphodiesterase I activities of the homogenate, are associated with lysosomes. After freezing-thawing, these activities remained essentially membrane-associated. The larger value obtained for alkaline phosphodiesterase I could not be ascribed to other lysosomal enzymes, as no such activity was detected at acidic pH. These two plasma membrane markers are thus unevenly distributed in the lysosomal compartment.     

Nitric oxide regulates the levels of cGMP accumulation in the cricket brain

Cricket brains were incubated in a saline containing nitric oxide (NO)-donor and phosphodiesterase inhibitor IBMX, which could activate soluble guanylate cyclase (sGC) to increase cGMP levels in the targets of NO. The increase of cGMP was detected by immunohistochemistry and enzyme linked immunosorbent assay. NO-induced cGMP immunohistochemistry revealed that many cell bodies of cricket brain showed cGMP immunoreactivity when preparations were treated with a saline containing 10 mM NO-donor SNP and phosphodiesterase inhibitor IBMX, but only a few cell bodies showed immunoreactivity when preparations were incubated without NO-donor. The concentration of cGMP in cricket brains were then measured by using cGMP-specific enzyme linked immunosorbent assay. Cricket brains were treated with a saline containing 1 microM of NO-donor NOR3 and 1 mM IBMX. The cGMP levels in the brain were increased about 75% compared to control preparations that was treated with a cricket saline containing IBMX. The level of cGMP decreased about 40% when preparations were incubated NOR3 saline containing sGC inhibitor ODQ. These results indicate that NO activates sGC and increases the levels of cGMP in particular neurons of the cricket brain and that the level of cGMP would be kept a particular level, which might regulate synaptic efficacy in the neurotransmission.     

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.     

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

Glucose transport in isolated brush-border and lateral-basal plasma-membrane vesicles from intestinal epithelila cells

Free-flow electrophoresis was used to separate microvilli from the lateral basal plasma membrane of the epithelial cells from rat small intestine. The activities of the marker enzyme for the microvillus membrane, i.e. alkaline phosphatase (EC 3.1.31), was clearly separated from the marker for the lateral-basal plasma membrane, i.e. the (Na⁺, K⁺)-ATPase (EC 3.6.1.3). A microvillus membrane fraction was obtained with a high specific activity of alkaline phosphatase (an 8-fold enrichement over the starting homogenate). The lateral-basal plasma membrane fraction contained (Na⁺, K⁺)-ATPase (5-fold over homogenate) with some alkaline phosphatase (2-fold over homogenate).Glucose transport was studied in both membrane fractions. The uptake of d-glucose was much faster than that of l-glucose in either plasma membrane, d-Glucose uptake could be accounted for completely by its transport into an osmotically active space. Interestingly, the characteristics of the glucose transport of the microvillus membrane were different from those of the lateral-basal plasma membrane. In particular: Na⁺ stimulated the d-glucose transport by the microvillus membrane, but not by the lateral-basal plasma membrane. In addition, the glucose transport of the microvillus membrane was much more sensitive to phlorizin inhibition than that of the lateral-basal plasma membrane.These experiments thus provide evidence not only for an asymmetrical distribution of the enzymes, but also for differences in the transport properties with respect to glucose between the two types of plasma membrane of the intestinal epithelial cell.     

Modulation of cardiac glycoside inhibition of (Na+ + K+)-ATPase by membrane lipids. Difference between species

The role of lipids in the modulation of the ouabain-sensitivity of membrane (Na+ + K+)-ATPase from different species has been studied using a reconstitution procedure which promotes lipid exchange during detergent depletion by Sephadex chromatography. Hybrid reconstitution of delipidated (Na+ + K+)-ATPase preparations from bovine brain into the lipids obtained from crab nerve enzyme preparations significantly reduces the sensitivity of the brain enzyme to inhibition by ouabain. Conversely, reconstitution of crab nerve enzyme into the lipids from bovine brain enzyme preparations increases the sensitivity of the crab enzyme to ouabain inhibition. These opposing effects demonstrate the role of membrane lipids in modulating the enzyme-inhibition relationship in preparations from these different species.     

Topography, purification and characterization of thyroidal NAD+ glycohydrolase

Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD(+) glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD(+) glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRT(l)) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD(+). The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD(+) glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-M(r) derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD(+) glycohydrolase activity of FRT(l) cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6mumol of nicotinamide released/h per mg). The purified enzyme displays a K(m) of 0.40mm for beta-NAD(+), a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent M(r) of 120000. Nicotinamide is an inhibitor (K(i) 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (K(i) 2.7mm). The purified enzyme splits beta-NAD(+), beta-NADP(+), beta-NADH and alpha-NAD(+) at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.     

Thyrotropin binding to and adenylate cyclase activity of porcine thyroid plasma membranes.

Plasma membranes have been purified from porcine thyroid gland homogenate by discontinuous sucrose gradient centrifugation. The preparations contained specific binding sites for thyrotropin but not for luteinizing hormone or the beta subunits of thyrotropin and luteinizing hormone. Optimum conditions of 125I-labeled thyrotropin binding were pH 6.0-6.5 and 37 degrees C. Thyrotropin binding was reduced by divalent (Ca2+, Mg2+) and monovalent cations (Na+, K+, Li+), 50% inhibition being obtained at 10 mM and 50 mM respectively. Displacement curves of 125I-labeled bovine or porcine thyrotropin by the unlabeled hormone from three species was in the order of increasing concentrations (bovine greater than porcine greater than human) which is the order of decreasing biological activity of these hormone preparations in the assay in vivo in the mouse. The validity of the results was established by controlling that porcine membranes bound the native and the 125I-labeled hormones with equal affinity. A single type of high-affinity (Kd = 0.28 nM) binding sites was detected for bovine and porcine thyrotropins. In contrast, porcine plasma membranes bound human thyrotropin with a lower affinity (Kd = 70 nM). A good correlation was found at equilibrium and in the conditions of the cyclase assay, between receptor occupancy and adenylate cyclase activation for the three hormones.     

Characteristics of a solubilized thyrotropin receptor from bovine thyroid plasma membranes.

The thyrotropin receptor from bovine thyroid plasma membranes has been solubilized using lithium diiodosalicylate, and an assay to measure thyrotropin binding to the solubilized receptor has been developed. Both the solubilized thyrotropin receptor and the thyrotropin receptor on thyroid plasma membranes have effectively identical nonlinear Scatchard plots and negatively sloped Hill plots, i.e. both preparations have receptors which appear to exhibit a similar negatively cooperative relationship. Although the pH optimum of thyrotropin binding to the solubilized receptor is the same as that of the thyroid plasma membrane receptor, pH 6.0, the pH dependency curve of the solubilized receptor is slightly different in its outline. Thyrotropin binding to the solubilized receptor is less sensitive to salt inhibition than is binding to the thyroid plasma membrane receptor; however, optimal binding remains at 0 degrees. The relative affinities of thyrotropin and two glycoprotein hormones which can be considered structural analogs, luteinizing hormone and human chorionic gonadotropin, are 100:10:5, respectively, toward plasma membrane receptors, but 100:25:40 toward the solubilized receptors. The solubilized receptor preparation is heterogeneous in size in that it has binding components with molecular weights of 286,000, 160,000, 75,000, and 15,000 to 30,000. Tryptic digestion converts all three higher molecular weight components to the 15,000 to 30,000 molecular weight species, and the 15,000 to 30,000 molecular weight receptor component has all of the binding properties of the solubilized receptor preparation before tryptic digestion including an identical nonlinear Scatchard plot. It has the same size as and coelutes from Sephadex G-100 with a 15,000 to 30,000 molecular weight receptor released by tryptic digestion of bovine thyroid plasma membranes or tryptic digestion of bovine or dog thyroid cells in culture. The tryptic fragment of the solubilized receptor or preparations has been purified almost 250-fold by affinity chromatography on thyrotropin-Sepharose columns. The binding activity is lost when the solubilized thyrotropin receptor preparation is exposed to beads of neuraminidase-Sepharose or conconavalin A-Sepharose.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. I. Biochemical determination

The distribution of adenylate cyclase (AC) in Golgi and other cell fractions from rat liver was studied using the Golgi isolation procedure of Ehrenreich et al. In liver homogenate the AC activity was found to decay with time, but addition of 1 mM EGTA reduced the rate of enzyme loss. The incorporation of 1 mM EGTA into the sucrose medium used in the initial two centrifugal steps of the Golgi isolation method stabilized the enzyme activity throughout the entire procedure and resulted in good enzyme recovery. In such preparations, AC activity was demonstrated to be associated not only with plasma membranes but also with Golgi membranes and smooth microsomal membranes as well. Furthermore, under the conditions used, enzyme activity was also associated with the 105,000 g x 90 min supernatant fraction. The specific activity of the liver homogenate was found to be 2.9 pmol-mg protein-1-min-1, the nonsedimentabel and microsomal activity was of the same order of magnitude, but the Golgi and plasma membrane activities were much higher. The specific activity of plasma membrane AC was 29 pmol-mg proten-1-min-1. The Golgi activity varied in the three fractions, with the highest activity (14 pmol) in GF1 lowest activity (1.8) in GF2, and intermediate activity (5.5) in GF3, when the Golgi activity was corrected for the presence of content protein, the activity in GF1 became much higher (9 x) than that of the plasma membrane while the activities in GF2 and GF3 were comparable to that of plasma membrane. In all locations studied, the AC was sensitive to NaF stimulation, especially the enzyme associated with Golgi membranes. The activities in plasma and microsomal membranes were stimulated by glucagon, whereas the Golgi and nonsedimentable AC were not.     

Possible role of regional superoxide dismutase activity and lipid peroxide levels in cadmium neurotoxicity: in vivo and in vitro studies in growing rats

Cd2+ (0.4 mg/kg) administration to growing rats (45 +/- 5 g) intraperitoneally, daily for 30 days was found to decrease the activity of superoxide dismutase (SOD) in all the brain regions, except hippocampus. The concentrations of lipid peroxides were significantly elevated in the cerebellum, cerebral cortex, corpus striatum and midbrain. A 100% inhibition in SOD activity was observed by 14 microM and 50 microM of Cd2+ in bovine blood and rat brain preparations, respectively. Cadmium-induced strong inhibitory effect on brain and purified bovine blood SOD suggested a direct effect of the metal on enzyme molecule. Furthermore, in vitro addition of a wide range of Cd2+ (1-100 microM) increased the rate of lipid peroxidation (LPO) reaction in fresh brain homogenate, however, did not affect boiled homogenate. The studies on LPO in reconstituted homogenate resulting from mixing of fresh and/or heated different subcellular fractions indicated the presence of some heat-labile Cd2+ -sensitive factor in 15000 x g pellet fraction. It is suggested that Cd2+ directly and indirectly through inhibition of SOD, increases the LPO of cell membranes and thus produces damage to the associated physiological functions leading to central nervous dysfunctions.     

Inhibition of phosphodiesterases relaxes detrusor smooth muscle via activation of the large-conductance voltage- and Ca²⁺-activated K⁺ channel

Detrusor smooth muscle (DSM) exhibits increased spontaneous phasic contractions under pathophysiological conditions such as detrusor overactivity (DO). Our previous studies showed that activation of cAMP signaling pathways reduces DSM contractility by increasing the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel activity. Here, we tested the hypothesis whether inhibition of phosphodiesterases (PDEs) can reduce guinea pig DSM excitability and contractility by increasing BK channel activity. Utilizing isometric tension recordings of DSM isolated strips and the perforated patch-clamp technique on freshly isolated DSM cells, we examined the mechanism of DSM relaxation induced by PDE inhibition. Inhibition of PDEs by 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor, significantly reduced DSM spontaneous and carbachol-induced contraction amplitude, frequency, duration, muscle force integral, and tone in a concentration-dependent manner. IBMX significantly reduced electrical field stimulation-induced contractions of DSM strips. Blocking BK channels with paxilline diminished the inhibitory effects of IBMX on DSM contractility, indicating a role for BK channels in DSM relaxation mediated by PDE inhibition. IBMX increased the transient BK currents (TBKCs) frequency by ～3-fold without affecting the TBKCs amplitude. IBMX increased the frequency of the spontaneous transient hyperpolarizations by ～2-fold and hyperpolarized the DSM cell resting membrane potential by ～6 mV. Blocking the BK channels with paxilline abolished the IBMX hyperpolarizing effects. Under conditions of blocked Ca(2+) sources for BK channel activation, IBMX did not affect the depolarization-induced steady-state whole cell BK currents. Our data reveal that PDE inhibition with IBMX relaxes guinea pig DSM via TBKCs activation and subsequent DSM cell membrane hyperpolarization.     

Iodide-induced inhibition of adenylate cyclase activity in horse and dog thyroid

The characteristics of the iodide-induced inhibition of cyclic AMP accumulation in dog thyroid slices have been previously described [Van Sande, J., Cochaux, P. and Dumont, J. E. (1985) Mol. Cell. Endocrinol. 40, 181-192]. In the present study we investigated the characteristics of the iodide-induced inhibition of adenylate cyclase activity in dog and horse thyroid. The inhibition of cyclic AMP accumulation by iodide in stimulated horse thyroid slices was similar to that observed in dog thyroid slices. The inhibition was observed in slices stimulated by thyroid-stimulating hormone, cholera toxin and forskolin. Increasing the concentration of the stimulators did not overcome the iodide-induced inhibition. Adenylate cyclase activity, assayed in crude homogenates or in plasma-membrane-containing particulates (100,000 x g pellets), was lower in homogenates or in particulates prepared from iodide-treated slices than from control slices. This inhibition was observed on the cyclase activity stimulated by forskolin, fluoride or guanosine 5'-[beta, gamma-imino]triphosphate, but also on the basal activity. It was relieved when the homogenate was prepared from slices incubated with iodide and methimazole. Similar results were obtained with dog thyroid. The inhibition persisted when the particulate fraction was washed three times during 1 h at 100,000 x g, in the presence of bovine serum albumin or increasing concentration of KCl. It was similar whatever the duration of the cyclase assay, in a large range of protein concentration. These results indicate that a stable modification of adenylate cyclase activity, closely related to the plasma membrane, was induced when slices were incubated with iodide. Iodide inhibition did not modify the affinity of adenylate cyclase for its substrate (MgATP), but induced a decrease of the maximal velocity of the enzyme. The percentage inhibition was slightly decreased when Mg2+ concentration increased, and markedly decreased when Mn2+ concentration increased. A detectable adenylate cyclase activity was demonstrated when intact slices were incubated in the presence of [alpha-32P]ATP, probably because of the presence of broken cells produced during the slicing. Iodide had no direct effect on this cyclase system, which confirms that iodide needs the integrity of the cell to induce the inhibition and suggests that the inhibition is not transmitted between cells.     

The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane.

To investigate the vital function(s) of the phosphoinositol-containing sphingolipids of Saccharomyces cerevisiae, we measured their intracellular distribution and found these lipids to be highly localized in the plasma membrane. Sphingolipids were assayed in organelles which had been uniformly labeled with [3H]inositol or 32P and by chemical measurements of alkali-stable lipid P, of long chain bases, and of very long chain fatty acids. We have developed an improved method for the preparation of plasma membranes which is based on the procedure of Duran et al. (Proc. Natl. Acad. Sci. USA 72:3952-3955, 1975). On the basis of marker enzyme and DNA assays carried out with a number of preparations, the plasma membranes contained less than 10% vacuolar membranes (alpha-mannosidase) and nuclei (DNA); the contamination by the endoplasmic reticulum (NADPH-cytochrome c reductase) varied from 0 to 20%. The plasma membrane preparations showed a 13-fold increase in the specific activity of vanadate-sensitive ATPase, compared with that in the homogenate, with a yield ranging from 50 to 80%. A comparison of the distribution of the ATPase with that of sphingolipids assayed by a variety of methods showed that 80 to 100% of the sphingolipids are localized in the plasma membrane; the sphingolipids constitute about 30% of the total phospholipid content of the plasma membrane. Minor amounts of sphingolipids that were found in isolated mitochondria and nuclei can be attributed to the presence of small amounts of plasma membrane in these fractions. These results suggest that one or more essential functions of these lipids is in the plasma membrane. Furthermore, sphingolipids may be useful chemical markers of the plasma membrane of S. cerevisiae.     

Subcellular structure of bovine thyroid gland. VII. A study on the distribution of bovine thyroid plasma membranes by density gradient centrifugation in zonal rotors.

In order to obtain plasma membrane-rich fractions two methods were tried. Approach A was based on differential pelleting followed by discontinous gradient centrifugation in a B-XIV zonal rotor. In approach B homogeneization was performed in buffered water (NaHCO3, pH 7.4). The 73 300 X g pellet from this homogenate was subjected to buoyant density equilibrium in a HS zonal rotor (continuous sucrose gradient). Using approach A, the highest relative specific activity for plasma membrane markers was found at the 30-37% sucrose interphase. However, an increase for glucose 6-phosphatase (endoplasmic reticulum marker) was also found at that interphase. Using approach B marker profiles different from approach A were found. Approach B results in a subdivision of membrane material in four distinct regions. These regions do not contain completely pure membrane species, although region I seems to be essentially derived from plasma membranes. It is also concluded from approach A that plasma membranes from bovine thyroid tissue are heterogeneous.     

Characterization, purification, and subcellular localization of bovine thyroid sialidases

Sialidase activities have been studied in bovine thyroid using sialoglycolipids, sialoglycoproteins, sialo-oligosaccharides and fluorogenic 4-methylumbelliferyl-alpha-D-N-acetylneuraminate as substrates. No sialidase activity could be detected towards native glycoprotein substrates. From enzyme kinetics, effector data and more convincingly from subcellular studies it became clear that in bovine thyroid at least two sialidase activities were present, a sialyllactitol sialidase confined to the lysosomal membrane and a glycolipid sialidase residing in the plasma membrane and displaying the features of a true ectoenzyme. The lipid requirement for full enzyme activity supported the membrane bound character of both sialidase activities. A soluble sialidase activity could not be demonstrated. After solubilization by CHAPS treatment, partial purification of the sialyllactitol sialidase could be achieved by affinity chromatography (Sepharose diamino dipropylamino-N-acetylneuraminic acid). The purified enzyme was extremely labile. Titration of the sialidase preparation with amino acid modifying agents revealed that sulfhydryl- and tryptophanyl groups were essential for the sialidase action.     

Calmodulin-binding proteins in granule and plasma membranes from bovine chromaffin cells

Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.     

Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.     

Subcellular Localization of Rat Brain Insulin Binding Sites

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, gamma-aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mitochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r = 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.