A Rapid Anterograde Axonal Transport of Carboxypeptidase H in Rat Sciatic Nerves

Abstract: Using the highly sensitive HPLC-fluorophotometry technique, anterograde and retrograde axonal transport of carboxypeptidase H (CPH), a putative pro-hormone processing enzyme that removes a basic amino acid from the C-terminus of a precursor peptide, was measured 12–72 h after double ligations of rat sciatic nerves. CPH-like activity in rat sciatic nerves was 60-fold lower than that in the pituitary gland. CPH-like enzyme activity was rapidly accumulated in the proximal segment and peaked 48 h after ligation. The axonal flow was 100 mm/day, indicating that CPH in rat sciatic nerves is rapidly transported to the nerve terminals as an active form. The properties of the enzyme were similar to those of CPH in the brain: The pH optimum is at 5.5, and the molecular mass is ∼50 kDa. These results suggest that active CPH in the PNS is transported by a rapid anterograde axonal flow and may play a role in converting proneuropeptides to active neuropeptides under the axonal transport.     

5-HYDROXY-l-TRYPTOPHAN DECARBOXYLASE ACTIVITY: MICROASSAY AND DISTRIBUTION IN DISCRETE RAT BRAIN NUCLEI

Abstract— 5-Hydroxy- l -tryptophan decarboxylase (EC 4.1.1.28) activity was measured in discrete regions of the rat brain by means of a sensitive micromethod lhat allows the quantitation of enzyme activity in μg amounts of brain tissue.
A good correlation was obtained between serotonin levels and 5-hydroxy- l -tryptophan decarboxylase activity in all the brain regions examined. Wide differences in enzyme activity were found in the different brain nuclei, with a 20-fold difference in activity between the most active region (the nucleus raphe dorsalis) and the least active regions (the corpus callosum and the optic tract).     

Binding of [3H]gamma-vinyl-GABA to GABA-transaminase in brain homogenates

[3H]Gamma-vinyl-GABA, an irreversible inhibitor of GABA-transaminase, was used to label the enzyme in homogenates of rat brain. The binding procedure utilized was found to be specific for GABA-transaminase and linear with tissue obtained from several regions of rat brain up to concentrations of 8 micrograms protein/microliter. The specific binding was directly proportional to the activity of the enzyme measured in vitro and was completely inhibited by the GABA-transaminase inhibitors aminooxyacetic acid (100 microM) and 3-mercaptopropionic acid (1.0mM). The binding procedure was used to estimate the amount of active enzyme present in a homogenate of striatal tissue.     

TWO FORMS OF ALANINE AMINOTRANSFERASE IN RAT BRAIN DURING ONTOGENY

Abstract— Alanine aminotransferase activity in subcellular fractions of rat brains was investigated during ontogenic development. The activity rose from the prenatal period until adulthood, the highest increase being observed during the period of morphological metabolic and functional maturation of the brain. The rise of the total activity was due predominantly to a rise in the activity of the cytosblic enzyme; the activity of the mitochondrial enzyme did not change markedly during ontogeny. CI-ions and elevated temperature (55°C) inhibited the activity only of the mitochondrial enzyme. Raised temperature stimulated the activity of the cytosolic enzyme while CI-ions did not influence its activity. Our results indicate that 2 alanine aminotransferase isoenzlmes are already present in the rat brain in the prenatal period. It is assumed that the cytosolic enzyme is involved in the regulation of tissue glycol)sis and alanine formation, while the mitochondrial enzyme plays a role in the amino nitrogen transport between mitochondria and cytosol.     

Calpain cleavage of brain glutamic acid decarboxylase 65 is pathological and impairs GABA neurotransmission

Previously, we have shown that the GABA synthesizing enzyme, L-glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2-3 times more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we examined the cleavage of fGAD65 under diverse pathological conditions including rats under ischemia/reperfusion insult as well as rat brain synaptosomes and primary neuronal cultures subjected to excessive stimulation with high concentration of KCl. We have shown that the formation of tGAD65 progressively increases with increasing stimulus concentration both in rat brain synaptosomes and primary rat embryo cultures. More importantly, direct cleavage of synaptic vesicle - associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme was detached from the synaptic vesicles. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate as indicated by microdialysis. Moreover, the levels of tGAD65 was also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain-mediated cleavage of fGAD65 is pathological, presumably due to decrease in the activity of synaptic vesicle - associated fGAD65 resulting in a decrease in the GABA synthesis - packaging coupling process leading to reduced GABA neurotransmission.     

Protein activator of cyclic 3':5'-nucleotide phosphodiesterase of bovine or rat brain also activates its adenylate cyclase.

Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol PX contained an activator which was separated from the enzyme by an anionic exchange resin column. Dissociation of the activator from adenylate cyclase rendered the enzyme less active, and reconstituting with an exogenous activator restored full enzyme activity. A pure protein activator of cyclic 3′:5′-nucleotide phosphodiesterase (EC 3.1.4.17) isolated from bovine brain also stimulated this adenylate cyclase. Stimulation of adenylate cyclase by the activator required Ca⁺⁺, the effect being immediate and reversible. Although the activator was specific, it lacked tissue specificity; an activator isolated from bovine brain cross-activated effectively adenylate cyclase from rat, and vice versa. These findings indicate that brain adenylate cyclase required an activator for activity and that this activator is functionally identical to the protein activator of phosphodiesterase (J.B.C. 249: 4943–4954, 1974).     

Estrone sulfate sulfohydrolase in the developing brain and pituitary of rat, mouse and guinea pig

The pattern of estrone sulfate sulfohydrolase (estrogen sulfatase) development in the brain of rat, mouse and guinea pig has been established by assaying whole homogenates. Activity was measurable in each species from the fetal state to adulthood. Maximum brain content was reached at about 20 days of age in rat, 14 days in mouse and 15 days in guinea pig. A considerable decrease occurred between 14 days and adulthood in mouse and lesser decreases were seen in rat and guinea pig. The subcellular distribution of enzyme in rat and mouse brain appeared to change from the immature to the adult state. No major differences in enzyme activity occurred between the sexes at any age. Tissue concentration of enzyme in the hypothalamic-preoptic area of rat and mouse was similar to that in the remainder of the brain. In guinea pig the brain concentration was slightly lower than that of the hypothalamic-preoptic region. Sulfatase content of the pituitary was low in all 3 species but the tissue concentration was considerably higher than that of brain, particularly in rat and mouse. Apparent Km values for brain sulfatase were in the range 6-17 microM, with no striking sex difference. Apparent Km's for pituitary sulfatase of immature rat and guinea pig were similar to those for brain in the same animals but that for mouse pituitary (0.9 microM) was much lower. It is unlikely that brain or pituitary sulfatase is by itself, a major factor in making available potentially active estrogen for use during differential sex development in these species.     

GALACTOCEREBROSIDE SULFOTRANSFERASE: FURTHER CHARACTERIZATION OF THE ENZYME FROM RAT BRAIN

Abstract— Myelin has an unusual lipid composition, being particularly rich in sulfatide. This lipid is synthesized by the transfer of sulfate from phosphoadenosine phosphosulfate to galactocerebroside, catalyzed by galactocerebroside sulfotransferase. This paper describes a sensitive assay for the sulfotransferase (capable of measuring activity in as little as 10 μg of extracted rat brain protein) so that this enzyme can be readily investigated in isolated cells, or the small amounts of tissue available in developing animals. Both manganase (20 m m ) and thiol reagents were required for optimal activity. This assay was used to monitor the purification of the sulfotransferase from rat brain. Extraction of the enzyme from crude homogenates required the nonionic detergent, Triton X-100, at pH 7–7.5. Removal of Triton X-100 from the extracted enzyme resulted in a soluble but less active enzyme, the activity of which could then be restored with detergents. Stability of the detergent-extracted enzyme was investigated, and even at —40°C there was a 20% loss of activity over 10 days. By standard procedures 500-fold purification of the enzyme has been achieved.     

Developmental changes of L-lysine-ketoglutarate reductase in rat brain and liver.

1. Developmental aspects of L-lysine-ketoglutarate reductase, the first enzyme in saccharopine pathway of L-lysine degradation in rat liver and brain tissues were studied. 2. Although the adult rat brain shows negligible activity, the enzyme activity was shown to be highly active during the early stages of development. 3. The enzyme activity gradually decreased through development in the brain, whereas it gradually increased in the liver, establishing the fact that the saccharopine pathway is the major pathway in liver. 4. Our results also show that glucagon stimulated the induction of this enzyme by 2-3-fold in both adult liver and brain tissues.     

Purification, molecular cloning, and genomic organization of human brain long-chain acyl-CoA hydrolase

An acyl-CoA hydrolase, referred to as hBACH, was purified from human brain cytosol. The enzyme had a molecular mass of 100 kDa and 43-kDa subunits, and was highly active with long-chain acyl-CoAs, e.g. a maximal velocity of 295 micromol/min/mg and K(m) of 6.4 microM for palmitoyl-CoA. Acyl-CoAs with carbon chain lengths of C(8-18) were also good substrates. In human brain cytosol, 85% of palmitoyl-CoA hydrolase activity was titrated by an anti-BACH antibody, which accounted for over 75% of the enzyme activity found in the brain tissue. The cDNA isolated for hBACH, when expressed in Escherichia coli, directed the expression of palmitoyl-CoA hydrolase activity and a 44-kDa protein immunoreactive to the anti-BACH antibody, which in turn neutralized the hydrolase activity. The hBACH cDNA encoded a 338-amino acid sequence which was 95% identical to that of a rat homolog. The hBACH gene spanned about 130 kb and comprised 9 exons, and was mapped to 1p36.2 on the cytogenetic ideogram. These findings indicate that the long-chain acyl-CoA hydrolase present in the brain is well conserved between man and the rat, suggesting a conserved role for this enzyme in the mammalian brain, and enabling genetic studies on the functional analysis of acyl-CoA hydrolase.     

DISTRIBUTION AND PROPERTIES OF ANGIOTENSIN CONVERTING ENZYME OF RAT BRAIN

Abstract— Angiotensin converting enzyme of rat brain was studied using Hip-His-Leu as substrate. The highest specific activity of the enzyme was associated with the microsomal fraction. The specific activity of the microsomal enzyme in several regions of the rat brain varied significantly. For example, the specific activities of the striatal and pituitary enzymes were about 10-fold greater than that of the cerebral cortical enzyme. The enzyme required chloride ion; moreover, activity was inhibited in the presence of disodium EDTA or O-phenanthroline, effects suggesting that the converting enzyme of brain is a metalloprotein. SQ-20881, a nonapeptide that inhibits converting enzyme in peripheral tissue, was a potent inhibitor of the enzyme of brain. In addition to Hip-His-Leu, the microsomal fraction was capable of liberating C terminal dipeptides from angiotensin I, Hip-Gly-Gly and Z-Gly- Gly-Val. The broad substrate specificity of the enzyme suggests that, in addition to the possible contribution of the enzyme to the brain renin-angiotensin system, other naturally occurring peptides might also be substrates for the enzyme.     

Tissue localization of 11 beta-hydroxysteroid dehydrogenase and its relationship to the glucocorticoid receptor.

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) dictates specificity for the mineralocorticoid receptor (MR) by converting the active steroid cortisol to cortisone in man (corticosterone to 11-dehydrocorticosterone in rodents), leaving aldosterone to occupy the MR. However cortisol is the principal circulating glucocorticoid in man and 11 beta-HSD, distributed in a tissue specific fashion, may represent a powerful mechanism in regulating exposure of active steroid to the glucocorticoid receptor (GR). A detailed localization study of 11 beta-HSD gene expression and activity in numerous rat tissues has been performed and compared with the presence of GR mRNA. 11 beta-HSD mRNA (1.4 kB) measured by hybridization to a cDNA derived from hepatic 11 beta-HSD, and enzyme activity, measured by percentage conversion of [3H]corticosterone to [3H]11-dehydrocorticosterone by tissue homogenate, was widespread, present in all tissues studied except spleen, brain cortex and heart. There was a close correlation between tissue 11 beta-HSD mRNA levels and activity (r = 0.91, P less than 0.001) suggesting pretranslational regulation of the enzyme at a tissue level. There was also close co-localization of GR mRNA (7 kB), measured by hybridization to a rat GR cRNA probe, and enzyme mRNA/activity in every tissue studied except heart and brain cortex in which GR mRNA was found. In the mineralocorticoid target tissues kidney and colon, additional 11 beta-HSD mRNA bands were seen (kidney 1.8 kB, colon 3.4 kB), suggesting the presence of multiple dehydrogenase species. 11 beta-HSD is widely distributed and suitably placed to modulate ligand occupancy of the GR. The possibility of multiple dehydrogenase species in mineralocorticoid target tissues is consistent with the hypothesis that the ubiquitous 'native' 1.4 kB hepatic enzyme regulates the GR, and these separate dehydrogenases regulate the MR.     

Retrograde alteration of 6-phosphogluconate dehydrogenase in axotomized superior cervical ganglia of the rat.

Mechanisms underlying increased activity of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase [decarboxylating] EC 1.1.1.44) in axotomized rat superior cervical ganglia were explored using a highly sensitive micro-immunochemical assay employing antibodies raised in rabbits against the purified enzyme. 6-Phosphogluconate dehydrogenase was purified from rat brain more than 1700-fold by salt fractionation, anion exchange, and immunoaffinity chromatography. The purified enzyme consisted of identical subunits having molecular weights of about 48,800 which could aggregate to catalytically active isomers of various sizes; however, only one form of the enzyme was detected in freshly prepared homogenates of rat neural tissue. Physical and immunological properties of the enzyme from rat brain were similar to those from superior cervical ganglia and liver. Augmented 6-phosphogluconate dehydrogenase activity noted in superior cervical ganglia 2 days after transection of major postganglionic nerve trunks was accompanied by a parallel increase in immunoreactive protein. Michaelis constants of the enzyme were the same in control and axotomized ganglia, and the presence of activators and inhibitors was not detected. It is concluded that increases in 6-phosphogluconate dehydrogenase subsequent to axotomy can be accounted for entirely by an increase in the steady state concentration of this protein.     

Studies on the Tissue Distribution of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases

An antiserum generated to the soluble form of the rat brain puromycin-sensitive enkephalin-degrading aminopeptidase was used to determine the tissue distribution of the soluble and membrane-associated forms of this enzyme. All tissues examined contained significant levels of the soluble enzyme form, with this enzyme accounting for greater than 90% of the arylamidase activity in brain, heart, and skeletal muscle. Native gel electrophoresis coupled with activity staining as well as inhibition studies were used to confirm the presence of this enzyme in various tissues. Serum was found not to contain this particular aminopeptidase. In contrast to the results obtained with the soluble enzyme form, brain was the only tissue found to contain the membrane-associated enzyme form. Although all tissues contained membrane-associated aminopeptidase activity only the brain enzyme could be maintained in solution in the absence of detergent. In addition, the brain membrane-associated enzyme could be distinguished from the membrane-associated aminopeptidase activity in other tissues on the basis of its sensitivity to inhibition by puromycin.     

Immunochemical characterization of a proline endopeptidase from rat brain. Its relationship to proline endopeptidase from other tissues and from other species

Monospecific antiserum raised against rat brain proline endopeptidase is used to demonstrate the ubiquity of the enzyme and its unique role in the degradation of proline-containing peptides. All endoproteolytic activity directed toward proline residues in several rat tissues is shown to share one or more common antigenic determinants with rat brain proline endopeptidase. Similar activity from tissue of other species crossreacts with rat proline endopeptidase. The data presented suggest that proline endopeptidase is the sole cytoplasmic enzyme capable of degrading proline-containing peptides in every tissue examined and that previously reported proline-specific endoproteolytic activities observed in a variety of systems may be ascribed to proline endopeptidase. The putative role of proline endopeptidase in protein degradation is discussed.     

ON THE MECHANISM OF STIMULATION OF AMP-AMINOHYDROLASE ACTIVITY BY HEXOKINASE IN BRAIN TISSUE

—A hexokinase has been isolated from brain tissue on Sephadex G-100 and DEAE cellulose which is similar to yeast enzyme in stimulating the AMP-aminohydrolase activity of rat brain soluble fractions. This effect of hexokinase is influenced neither by N-acetyl-glucosamine nor noradrenaline. An isoenzyme of hexokinase isolated from brain tissue on DEAE cellulose, having properties similar to that of the muscle enzyme, has no effect on AMP-aminohydrolase activity. The activating effect of yeast hexokinase is not due to its oligomeric structure. Enzyme subunits obtained by the treatment of native yeast enzyme by urea also activate AMP-aminohydrolase of rat brain soluble fractions.     

Effects of different components of Mao Dongqing’s total flavonoids and total saponins on transient ischemic attack (TIA) model of rats

Objective: To study the effects of the different components of the total flavonoids and total saponins from Mao Dongqing's active site on the rats of TIA model, determine the optimal reactive components ratio of Mao Dongqing on the rats of TIA. Methods: TIA rat model was induced by tail vein injection of tert butyl alcohol, the blank group was injected with the same amount of physiological saline, then behavioral score wasevaluated.Determination the level of glutamic acid in serum, the activity of Na+-K+-ATP enzyme, CA⁺⁺-ATP enzyme and Mg⁺⁺-ATP enzyme in Brain tissue, observe the changes of hippocampus in brain tissue, the comprehensive weight method was used to evaluate the efficacy of each component finally. Results: The contents of total flavonoids and total saponins in the active part of Mao Dongqing can significantly improve the pathological changes of brain tissue in rats, improve the activity of Na⁺-K⁺-ATP enzyme, Ca⁺⁺-ATP enzyme and Mg⁺⁺-ATP enzyme in the brain of rats, and reduce the level of glutamic acid in serum. The most significant of the contents was the ratio of 10:6. Conclusion: The different proportions of total flavonoids and total saponins in the active part of Mao Dongqing all has a better effect on the rats with TIA, and the ratio of 10:6 is the best active component for preventing and controlling TIA.     

Microsomal phospholipase D of rat brain and lung tissues

Phospholipase D of rat brain and lung tissue is enriched in the microsomal fraction. A comparative study of the activity of this enzyme from various rat tissues revealed that significantly greater levels were present in brain and lung particles than in other tissues assayed. The enzyme was found to have an absolute requirement for detergent and a pH optimum of 6.5. This is the first report of the detection of membrane bound phospholipase D activity.     

Subcellular distribution of diacylglycerol lipase in rat and mouse brain

Rat Brain has a lipase which hydrolyzes diacylglycerol at an optimal pH of 4.8 (1). The subcellular distribution of this acid diacylglycerol lipase was studied in brain tissue of rats and mice; in the latter case neurological mutants and their normal controls were used. Several other acidic hydrolases were employed as normal controls were used. Several other acidic hydrolases were employed as lysosomal markers. In mouse brain, the specific activity which is about 50-100 times lower than in rat brain, was greatest in the lysosomal fraction. In contrast, no enrichment of DG-lipase was observed in any subcellular fraction of the active enzyme of rat brain. Activities were about equally distributed in the microsomal, myelin-synaptosomal and lysosomal fractions.