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.     

Parvalbumin in Cat Brain: Isolation, Characterization, and Localization

Because of the increasing evidence that Ca2+-binding proteins have important regulating functions in nerve cells and because of the indications that there are species differences in the structures of these proteins, parvalbumin was purified from cat brain and muscle. Brain and muscle parvalbumins were found to be indistinguishable from each other in their biochemical and immunological properties. However, cat parvalbumin differs from all other mammalian parvalbumins by its apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 10-11K (compared to rat parvalbumin, 12K), and a lower pI of 4.6 (rat parvalbumin, 4.9), in the tryptic peptide maps, and in the immunological properties, indicating a distinct primary structure. With the purified parvalbumin as antigen, polyclonal antibodies were raised in rabbits and these were subsequently used for immunohistochemical localizations of parvalbumin in the cat brain. In the visual cortices of adult cats immunoreactive neurons were present throughout layers II and IV. In cerebellar cortex, Purkinje, basket, and stellate cells were immunoreactive. Comparison with staining patterns obtained with antiserum against rat parvalbumin revealed some cross-reactivity but confirmed the existence of species differences in the antigenic structure of rat and cat parvalbumin.     

l-Histidine Decarboxylase in the Human Brain: Properties and Localization

The properties of the histamine-forming enzyme in human brain samples were studied utilizing a radiochromatographic procedure. The influence of postmortem conditions was checked with rat brains, and the results indicated that the enzyme activity is not altered in situ for a delay not exceeding 4 h at ambient temperature. Moreover, tissue blocks or homogenates can be stored at low temperatures for up to 3 months with a good preservation of the enzyme activity. The data indicate that histamine synthesis in the human brain involves the ?specific” histidine decarboxylase (HD, EC 4.1.1.22) and not the aromatic l -amino acid decarboxylase; (1) the optimum pH is 7.4 at 10^-6m-l -histidine; (2) the apparent K_m is about 3.10^-5m ; (3) it is inhibited by α-hydrazino histidine and brocresine but not affected by α-methyl DOPA. Moreover, a major portion of the enzyme is localized in a subcellular fraction containing nerve terminals and it shows an uneven regional distribution which parallels that observed in the brain of other mammalian species. Taken together these data strongly suggest that histamine could play a neurotransmitter role in the human brain.     

Glutathione in Brain: Overview of Its Conformations,Functions, Biochemical Characteristics,Quantitation and Potential Therapeutic Role in Brain Disorders

Neurochemical Research - Glutathione (GSH) is an important antioxidant found abundantly and synthesized intracellularly in the cytosol in a tightly regulated fashion. It has diverse physiological...     

Purification, Characterization, and Localization of Aspartoacylase from Bovine Brain

Canavan disease, an autosomal recessive disorder, is characterized biochemically by N-acetylaspartic aciduria and aspartoacylase (N-acyl-L-aspartate amidohydrolase; EC 3.5.1.15) deficiency. However, the role of aspartoacylase and N-acetylaspartic acid in brain metabolism is unknown. Aspartoacylase has been purified to apparent homogeneity with a specific activity of approximately 19,000-20,000 nmol of aspartate released/mg of protein. The native enzyme is a 58-kDa monomer. The purified aspartoacylase activity is enhanced by divalent cations, nonionic detergents, and dithiothreitol. Low levels of dithiothreitol or beta-mercaptoethanol are required for enzyme stability. Aspartoacylase has a Km of 8.5 x 10(-4) M and a Vmax of 43,000 nmol/min/mg of protein. Inhibition of aspartoacylase by glycyl-L-aspartate and amino derivatives of D-aspartic acid suggests that the carbon backbone of the substrate is primarily involved in its interaction with the active site and that a blocked amino group is essential for the catalytic activity of aspartoacylase. Biochemical and immunocytochemical studies revealed that aspartoacylase is localized to white matter, whereas the N-acetylaspartic acid concentration is threefold higher in gray matter than in white matter. Our studies so far indicate that aspartoacylase is conserved across species during evolution and suggest a significant role for aspartoacylase and N-acetylaspartic acid in normal brain biology.     

Mitochondrial Malic Enzyme: Purification from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization in Neurons of Rat Brain

Abstract: To elucidate the cellular location of mitochondrial malic enzyme in brain, immunocytochemical studies were performed. For this purpose, mitochondrial malic enzyme was purified to apparent homogeneity from bovine brain and used for the immunization of rabbits. Subjecting the antiserum to affinity purification on immobilized antigen as an absorbent yielded a purified immunoreactive antibody preparation, which was characterized by probing cytosolic and mitochondrial fractions of bovine and rat brain in western blotting. As neither crossreactivity with cytosolic malic enzyme nor immunoreactivity against other proteins could be observed, the antibody preparation was found suitable for immunocytochemistry. By using sections of perfusion-fixed rat brain, considerable resolution was achieved at the light-microscopic level. Distinct and specific staining of neurons was observed; in contrast, no staining of astrocytes and possibly unspecific staining within the nuclei of oligodendrocytes were obtained. From these data, it is concluded that mitochondrial malic enzyme is located in neurons; however, in astrocytes, the enzyme appears to be either lacking or present at a much lower level. A protective role against oxidative stress in neurons is proposed for mitochondrial malic enzyme.     

Localization of Glycogen Synthase in Brain

Antisera against glycogen synthase from canine brain were prepared and used for investigation of the localization of the enzyme in the brain. Antisera cross-reacted only with the 88-kilodalton protein that is the subunit of brain glycogen synthase. Immunoreactivity of glycogen synthase was universally distributed in all regions of the brain, although hippocampus, cerebral cortex, caudatoputamen, and cerebellar cortex had relatively high immunoreactivity. Light microscopic examination revealed that the immunoreactivity was found in all cell types, such as neurons in several regions, astrocytes, ependymal cells surrounding the ventricle, oligodendrocytes, and epithelial cells of the choroid plexus in the ventricle. Immunoreactive intensity was more prominent in neurons than glial cells. Immunostaining may be a useful tool for investigation of the state of glycogen metabolism under normal and pathological conditions.     

Brain Insulin Dysregulation: Implication for Neurological and Neuropsychiatric Disorders

Arduous efforts have been made in the last three decades to elucidate the role of insulin in the brain. A growing number of evidences show that insulin is involved in several physiological function of the brain such as food intake and weight control, reproduction, learning and memory, neuromodulation and neuroprotection. In addition, it is now clear that insulin and insulin disturbances particularly diabetes mellitus may contribute or in some cases play the main role in development and progression of neurodegenerative and neuropsychiatric disorders. Focusing on the molecular mechanisms, this review summarizes the recent findings on the involvement of insulin dysfunction in neurological disorders like Alzheimer’s disease, Parkinson’s disease and Huntington’s disease and also mental disorders like depression and psychosis sharing features of neuroinflammation and neurodegeneration.     

Unique Features of the Insulin Receptor in Rat Brain

We examined the structure of the affinity-labeled insulin receptors in rat brain, rat liver, and human IM-9 lymphocytes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In gels run under reducing conditions, the alpha-subunit of the insulin receptor in brain had an apparent Mr of 127,000 distinctly lower than that seen in both rat liver and human lymphocytes (apparent Mr = 136,000). Exposure to neuraminidase increased the electrophoretic mobility of the liver receptor, but had no effect on the insulin receptor in brain. The carbohydrate moieties of the insulin receptors in rat brain and liver were further examined by chromatography on wheat-germ agglutinin agarose. The receptors in both tissues adsorbed to the wheat-germ agglutinin; elution with 0.3 M N-acetyl glucosamine resulted in slightly better recovery of the brain than of the liver receptor. Exposure to neuraminidase virtually abolished the interaction of the liver receptor with the lectin, whereas adsorption of the brain receptor was unaffected by neuraminidase. These results indicate that the insulin receptor in brain is distinguished from those in peripheral tissues by structural alterations, including changes in the carbohydrate moiety of the receptor. Such alterations contrast sharply with the previously observed similarities in insulin binding properties between insulin receptors in brain and other tissues. The implications of such structural alterations for the program of insulin action expressed by the receptors in brain remain to be explored.     

Development and Localization of the Thymidine Phosphorylating Systems in Brain

Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[³H]thymidine at 37°C under 95% O₂-5% CO₂. When slices from all brain regions of 2-day-old rabbits were incubated in [³H]thymidine for 30 min, tissue-to-medium ratios of ³H were between 2 and 4 and declined with age, and the percentages of the total ³H in perchloric acid homogenates of brain slices as [³H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the ³H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [³H]thymidine for 30 min, the highest percentage of [³H]thymidine phosphates plus [³H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [³H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [³H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span.     

Properties and Localization of the Sulfate-Activating System in Rat Brain

The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin Binding Sites in Human and Monkey Brain: Localization and Characterization

A radioiodinated analogue of somatostatin 28, 125I [Leu8,D-Trp22,Tyr25] SS-28, was used to localize and characterize somatostatin binding sites in both human and monkey brain. High-affinity binding sites (approximately 1 nM) were found in cerebral cortex. The highest binding was in cerebral cortex with intermediate binding found in hippocampus, striatum, and amygdala and low binding in hypothalamus and brainstem. There was a rough correlation between somatostatin receptor binding and concentrations of somatostatin-like immunoreactivity (SLI) in human brain. Somatostatin receptors were stable for up to 24 h in an animal model simulating human autopsy conditions and there was no correlation between postmortem interval and receptor binding in human brain. Pharmacologic characterization in human cortex showed that there was a correlation between the inhibition of receptor binding by somatostatin analogues and their known abilities to inhibit growth hormone secretion. These findings demonstrate that a highly specific membrane-associated receptor for somatostatin is present in both monkey and human brain. Examination of somatostatin receptor binding in Alzheimer's disease and Huntington's disease may improve understanding of the role of somatostatin in both these illnesses.     

Superoxide Dismutases of Escherichia coli: Intracellular Localization and Functions

Escherichia coli B contains two superoxide dismutases which differ with respect to their localization within the cell, the nature of their prosthetic metals, their responses to changes in (p)O(2), and their functions. One of these enzymes, which was liberated from the cells by osmotic shock and which was therefore presumed to be localized in the periplasmic space, is an iron-containing superoxide dismutase. The amount of this iron enzyme did not vary in response to changes in (p)O(2) during growth. In contrast, the other superoxide dismutase was not solubilized by osmotic shock, was a mangano-protein, and was found in greater amounts in cells which had been grown at high (p)O(2). E. coli, which had low levels of the iron-enzyme and high levels of the mangano-enzyme, as a consequence of growth in iron-deficient aerated medium, was killed by exposure to an exogenous flux of O(2) (-) which was generated either photochemically or enzymatically. The addition of bovine superoxide dismutase to the suspending medium protected these cells against this stress. On the other hand, E. coli, which had high levels of the iron-enzyme and low levels of the mangano-enzyme, as a consequence of growth in iron-rich anaerobic medium, was resistant to exogeneous O(2) (-). On the basis of these and of previously reported results (4a, Yost, F. J. and I. Fridovich, J. Biol. Chem., 1973, in press), it appears that the iron superoxide dismutase, of the periplasmic space, serves as a defense against exogenous O(2) (-), whereas the mangano-superoxide dismutase, in the matrix of these cells, serves to counter the toxicity of endogenous O(2) (-).     

Human Brain Phenol Sulfotransferase: Biochemical Properties and Regional Localization

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenol and catechol drugs. The human blood platelet contains a thermolabile (TL) form of PST that catalyzes the sulfate conjugation of dopamine and other monoamines and a thermostable (TS) form that catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol. Experiments were performed to determine whether the brain contains forms of PST analogous to the TL and TS forms found in the human platelet, and to determine whether there are regional variations in human brain PST activity. We found that the human brain contains at least two forms of PST, forms that are similar to the platelet TS and TL forms of the enzyme with respect to substrate specificity, apparent Km constants, thermal stability, and sensitivity to inhibitors. Optimal conditions were determined for the measurement of these two activities in brain homogenates. The stability of PST activities in the human brain after death was determined in five samples of cerebral cortex that were obtained during clinically indicated neurosurgical procedures. An average of 76 +/- 8% and 80 +/- 9% (mean +/- SEM) of the basal TL and TS PST activities, respectively, remained in these five samples of cerebral cortex after 8 h of storage under simulated post-mortem conditions. Six human brains were then obtained less that 8 h after death from patients who had no neurological disease prior to death. The mean activities of the TL and TS forms of PST were measured in 17 different regions of the six brains. If the pituitary was excluded from consideration, TL and TS PST activities both varied approximately fivefold among these regions, and both activities were highest in cerebral cortex. However, the average TS activity in the anterior pituitary, a tissue of non-neural origin embryologically, was 6.5-fold greater than the highest average TS PST activity found in cerebral cortex.     

The Role of Insulin C-Peptide in the Coevolution Analyses of the Insulin Signaling Pathway: A Hint for Its Functions

As the linker between the A chain and B chain of proinsulin, C-peptide displays high variability in length and amino acid composition, and has been considered as an inert byproduct of insulin synthesis and processing for many years. Recent studies have suggested that C-peptide can act as a bioactive hormone, exerting various biological effects on the pathophysiology and treatment of diabetes. In this study, we analyzed the coevolution of insulin molecules among vertebrates, aiming at exploring the evolutionary characteristics of insulin molecule, especially the C-peptide. We also calculated the correlations of evolutionary rates between the insulin and the insulin receptor (IR) sequences as well as the domain-domain pairs of the ligand and receptor by the mirrortree method. The results revealed distinctive features of C-peptide in insulin intramolecular coevolution and correlated residue substitutions, which partly supported the idea that C-peptide can act as a bioactive hormone, with significant sequence features, as well as a linker assisting the formation of mature insulin during synthesis. Interestingly, the evolution of C-peptide exerted the highest correlation with that of the insulin receptor and its ligand binding domain (LBD), implying a potential relationship with the insulin signaling pathway.     

Characterization and Regulation of Insulin Receptors in Rat Brain

An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD = 3-11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four-fold greater (Bmax = 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 microM insulin for 3 h at 37 degrees C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner.     

Distribution and Localization of Estrogen-Sensitive Dopamine Receptors in the Rat Brain

Administration of estrogen to adult male rats increases the density of striatal dopamine receptors. The densities of the dopamine receptors in the nucleus accumbens and cortex are not altered, while the density of those in the hippocampus is decreased. In the pituitary the density, on a whole pituitary basis, is not changed. The increased density of striatal dopamine receptors normally observed after estrogen treatment is prevented by prior injection into the striatum of kainic acid, which destroys the intrinsic neurons in the striatum. In addition, the benzodiazepine receptors in the striatum, cortex, hippocampus, and cerebellum are not altered by estrogen treatment, showing the specificity of the estrogen treatment and suggesting that the effects of estrogen are not mediated through benzodiazepine receptors.     

Insulin Binding in Four Regions of the Developing Rat Brain

Specific insulin binding has been demonstrated in partially purified membranes prepared from four regions of the developing rat brain. Insulin binding to brain membranes demonstrated kinetics and hormonal specificity that were quite similar to those reported for traditional insulin target tissues (e.g., liver and adipose tissue), and binding was significantly correlated with receptor concentration. Binding in the olfactory bulbs, cerebrum, cerebellum, and hypothalamus all reached highest values at 15 days of postnatal life, with the olfactory bulbs generally showing the greatest binding at all ages studied. A temporal relationship was found between insulin binding to brain membranes in the postnatal rat and plasma membrane protein synthesis, especially in the cerebellum and olfactory bulbs.