Metabolic pathways for the degradation of phosphatidic acid in isolated nuclei from cerebellar cells.

The aim of the present research was to analyse the pathways for phosphatidic acid metabolism in purified nuclei from cerebellar cells. Lipid phosphate phosphatase and diacylglyceride lipase activities were detected in nuclei from cerebellar cells. It was observed that DAGL activity makes up 50% of LPP activity and that PtdOH can also be metabolised to lysophosphatidic acid. With a nuclear protein content of approximately 40 µg, the production of diacylglycerol and monoacylglycerol was linear for 30 min and 5 min, respectively, whereas it increased with PtdOH concentrations of up to 250 µM. LysoPtdOH, sphingosine 1-phosphate and ceramide 1-phosphate, which are alternative substrates for LPP, significantly reduced DAG production from PA. DAG and MAG production increased in the presence of Triton X-100 (1 mM) whereas no modifications were observed in the presence of ionic detergent sodium deoxycholate. Ca²+ and Mg²+ stimulated MAG production without affecting DAG formation whereas fluoride and vanadate inhibited the generation of both products. Specific PtdOH-phospholipase A1 and PtdOH-phospholipase A2 were also detected in nuclei. Our findings constitute the first reported evidence of active PtdOH metabolism involving LPP, DAGL and PtdOH-selective PLA activities in purified nuclei prepared from cerebellar cells.

Subcellular distribution and properties of guanylate cyclase in rat cerebellum.

In rat cerebellum the major portion of guanylate cyclase was found to be particulate-bound. The properties of particulate and supernatant guanylate cyclases from the cerebellum were comparatively examined. Both enzymes required the same optimal concentration of Mn2+ and were stimulated by Ca2+ in the presence of a low concentration of Mn2+. But dispersion of the particulate enzyme with Triton X-100 altered the Mn2+ concentration producing maximum activity and the inhibitory effect of Ca2+. The subcellular distributions of guanylate and adenylate cyclases were also studied in rat cerebellum. The major portions of the two cyclases were found in the mitochondrial fraction. The submitochondrial fractions separated by sucrose gradient showed that the major activities of both cyclases were concentrated in the fraction containing mainly nerve ending particles.

Regulation of a presynaptic adenylate cyclse from bovine cerebellum by beta-adrenergic receptors.

Intact crude synaptosome from bovine cerebellum contain, in addition to an externally accessible (postsynaptic) adenylate cyclase, an enzyme with its catalytic center oriented towards the inside of the synaptosome (presynaptic adenylate cyclase). This is demonstrated by the unmasking of latent adenylate cyclase activity by Triton X-100. Furthermore, intact crude synaptosomes can synthesize cyclic AMP from adenine. This synthesis takes place inside the synaptosomes as the postsynaptic adenylate cyclase is inactive in the Krebs-Ringer buffer. Presynaptic adenylate cyclase activity is not influenced by depolarization, as shown by [3H]adenine pulse-labeling, but is stimulated by (-)-norepinephrine and (-)-isoproterenol. (+/-)-Propranolol inhibits this stimulation whereas phentolamine has no effect, suggesting the presence of a beta-adrenergic receptor-coupled presynaptic adenylate cyclase.

Enhanced resolution of histochemical distribution of glucose-6-phosphate dehydrogenase activity in rat neural tissue by use of a semipermeable membrane.

We examined the histochemical distribution of glucose-6-phosphate dehydrogenase (G6PD) activity in neural tissue using different diffusion barriers. Although polyvinyl alcohol and agar overlays permitted regional localization of G6PD, a semipermeable membrane revealed cellular differences in G6PD activity within populations of neurons. Distribution of G6PD activity in selected regions of the nervous system was examined using the membrane technique. White matter usually exhibited strong G6PD activity. The neuronal somata of the dorsal root ganglia (L4-L6) and anterior horns of the spinal lumbar enlargement demonstrated a variation in activity which was independent of somal size. Satellite cells showed intense activity when the membrane technique was used. Hippocampal pyramidal and granular cells of the dentate gyrus exhibited moderate, uniform G6PD activity, but only weak activity was seen in hippocampal and dentate molecular layers. High levels of activity were observed in the vascular endothelial cells of the brain, spinal cord, and choroid plexus, and in the ependymal cells of the spinal central canal and ventricles of the brain. The superior vestibular nucleus appeared to have little G6PD activity in either the neuron cell bodies or the surrounding parenchyma. The use of a semipermeable membrane for localization of G6PD activity in neural tissues permits enhanced resolution of neuron elements and may provide a more accurate assessment of G6PD activity in histological preparations.

Purification of insoluble nitric oxide synthase from rat cerebellum.

Nitric oxide synthase [EC 1.14.23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100,000 x g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2',5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.

Mitochondrial fusion protects against neurodegeneration in the cerebellum.

Mutations in the mitochondrial fusion gene Mfn2 cause the human neurodegenerative disease Charcot-Marie-Tooth type 2A. However, the cellular basis underlying this relationship is poorly understood. By removing Mfn2 from the cerebellum, we established a model for neurodegeneration caused by loss of mitochondrial fusion. During development and after maturity, Purkinje cells require Mfn2 but not Mfn1 for dendritic outgrowth, spine formation, and cell survival. In vivo, cell culture, and electron microscopy studies indicate that mutant Purkinje cells have aberrant mitochondrial distribution, ultrastructure, and electron transport chain activity. In fibroblasts lacking mitochondrial fusion, the majority of mitochondria lack mitochondrial DNA nucleoids. This deficiency provides a molecular mechanism for the dependence of respiratory activity on mitochondrial fusion. Our results show that exchange of mitochondrial contents is important for mitochondrial function as well as organelle distribution in neurons and have important implications for understanding the mechanisms of neurodegeneration due to perturbations in mitochondrial fusion.

Synthesis of phosphatidylserine by base exchange in Triton-insoluble floating fractions from rat cerebellum.

Phosphatidylserine (PS), which is synthesized in mammalian tissues by the exchange between free serine and the nitrogen bases present in membrane glycerophospholipids, is strictly required for protein kinase C (PKC) activity. PKC, as other molecules involved in signal transduction, is present in lipid rafts, considered as a platform for molecular signaling. Membrane microdomains enriched in components of rafts can be isolated on the basis of their insolubility in Triton X-100 at 4 degrees C and their low density in sucrose density gradient. This study demonstrates the existence of serine base exchange enzyme (SBEE) in Triton-insoluble floating fractions containing associated PKC. Using two fractions of detergent-resistant membranes from rat cerebellum, we observed a correlation between the level of SBEE activity and that of membrane-associated PKC. This suggests that SBEE, synthesizing PS in the binding area for PKC, participates to signal transduction. The capability of SBEE to utilize not only serine but also ethanolamine, as free exchanging base, suggests a mechanism for modulating in loco PS concentration.

A comparison of the ATP: L-methionine-S-adenosyltransferase of rat cerebral cortex and cerebellum.

The ATP: L-methionine-S-adenosyltransferase of rat cerebral cortex and cerebellum was found to be differentially responsive to solubilization by sodium deoxycholate. Furthermore, the cerebellar enzyme was markedly less sensitive to inactivation by deoxycholate and to storage at 4 degrees C. The specific activity of the cerebellar enzyme was significantly higher and the two enzyme activities also exhibited differences in apparent Km values for L-methionine.

Possible regulation of hypothalamus and lung histidine decarboxylase activity by cAMP-dependent protein kinase.

Activity of crude histidine decarboxylases (HisDC) from the hypothalamus and the lungs, was markedly reduced by incubating with ATP.Mg, cAMP and cAMP-dependent protein kinase A, whereas activity of the crude glandular stomach enzyme changed only slightly under equal condition. The omission of one of these components failed to reduce HisDC activity by as much as the complete system. Addition of bovine heart (type II) or rat cerebellum protein kinase A (types I and II) inhibitor to the assay prevented enzyme inactivation; moreover, protein kinase A inhibitors permitted moderate activation under phosphorylating and control conditions. Cytosolic hypothalamus HisDC activity was elevated 2-2.2-fold by incubating the cytosol for 15 min in the presence of MnCl2, a known stimulator of phosphoprotein phosphatase; this was prevented when 20 mM NaF, a common inhibitor of phosphoprotein phosphatase, was added to the cytosol. The apparent Km of ATP.Mg-treated hypothalamus HisDC for histidine was elevated 5-10-fold compared to controls, whereas the Vmax was approximately the same. Under this condition, the Km was calculated as high as 0.5-2.2 mM (depending on phosphorylating conditions), while controls had a Km of 0.1-0.3 mM (depending on the initial phosphorylating states). Addition of rabbit muscle (type I), bovine heart (type II) or rat cerebellum (types I and II) inhibitor of protein kinase A, to the phosphorylating mixture, abolished the difference in Km between control and ATP.Mg-treated HisDC. Moreover, rat cerebellum protein kinase A inhibitors increased Vmax to above the control level; while 20 mM NaF (inhibitor of phosphoprotein phosphatase) decreased Vmax to approximately one half of that of the controls. These data indicate that HisDC activity in the hypothalamus and the lungs, but not in the stomach, is affected in oppositely by protein kinase A and phosphoprotein phosphatases.

Appearance of myelin proteins during development in the chick central nervous system.

The time course of the appearance of myelin-specific markers was studied in the developing chick central nervous system (CNS). Chick CNS tissue was studied for the presence of both proteolipid and myelin basic protein by electroblotting and for 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) by enzyme assay. Four regions of chick spinal cord (cervical, brachial, thoracic and lumbar), brain stem, cerebellum, optic nerve and cortex were studied. In general, myelin basic protein appeared approximately 1 day earlier than proteolipid. In spinal cord and brain stem, myelin basic protein appeared at 13 days incubation. In cerebellum and optic nerve, it appeared at 17 days incubation and in cortex at hatching. CNPase activity increased in most CNS regions between 16 days incubation and hatching. These results suggest that myelination occurs earlier in the chick than in the rat and that it occurs over a shorter time period.

Identification of an ectokinase activity in cerebellar granule primary neuronal cultures.

Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-32P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for approximately 20 min. Unlike what was seen with [gamma-32P]ATP, in the presence of [32P]orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80-90% in the presence of 1 microM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO4(3-). Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 microM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.

The effect of nonionic detergents on the activity and/or stability of rat brain nitric oxide synthase.

The results of this study indicate that the addition of low concentrations of a nonionic detergent such as those represented by the Tween, Brij, or Triton classes causes an apparent activation of nitric oxide synthase. It is possible that this apparent activation is due to the ability of these detergents to stabilize the protein. The stabilizing influence of the detergents may be a result of inhibiting the dissociation of the dimeric protein into monomers or the dissociation of an essential cofactor or prosthetic group from the active enzyme. Regardless of the mechanism of action, the addition of low concentrations of nonionic detergents results in longer and increased nitric oxide synthase activity and may be an important tool for those involved in enzymological studies of nitric oxide synthase.

Some kinetic and chromatographic properties of detergent-dispersed adenylate cyclase.

Studies on the reaction kinetics and chromatographic properties of detergent-dispersed adenylate cyclase are described. Detergent-dispersed enzyme was prepared from whole rat cerebellum and from partially purified plasma membranes from rat liver. Data were simulated to fit kinetic models for which an inhibitor is added in constant proportion to the variable substrate. Models were chosen to distinguish whether the adenylate cyclase reaction may be controlled by an inhibitory action of free ATP--4 (or HATP--3) or by a stimulatory action of free divalent cations. The various kinetic models were then tested with the dispersed brain adenylate cyclase with both Mg++ and Mn++ and in two different buffer systems. The experimental data indicate that this enzyme has a distinct cation binding site, but exhibits no significant inhibition by HATP--3 or ATP--4. The detergent-dispersed adenylate cyclase both from liver plasma membranes and from brain have been chromatographed on anion exchange material and have been subjected to gel filtration. The presence of detergent was required for elution of cyclase activity from DEAE-Sephadex but was not required when DEAE-agarose was used. Dispersed brain cyclase was also chromatographed on agarose-NH(CH2)3NH(CH2)3-NH2 which exhibits both ionic and hydrophobic properties. Fifty percent of the applied activity was recovered with a fivefold increase in specific activity. The data suggest that the relative effectiveness of a given chromatographic procedure for detergent-dispersed adenylate cyclase may reflect the influence of both hydrophobic and ionic factors.