THE PRESENCE OF AN α-GALACTOSIDASE IN BRAIN AND ITS ROLE IN THE DEGRADATION OF THE DIGALACTOSYL DIGLYCERIDE OF BRAIN

Abstract— The presence of α-galactosidase activity has been demonstrated in rat brain. This enzyme, located mainly in the crude mitochondrial fraction, actively hydrolysed the substrates p -nitrophenyl-α-galactoside and melibiose, and also catalysed the hydrolysis of digalactosyl diglyceride of both animal and plant origin. The hydrolysis of p -nitrophenyl-α-galactoside, as catalysed by the α-galactosidase, occurred optimally at pH 4·9, showed an approximate K _m of 1·0 × 10⁻³ m , and was markedly inhibited by melibiose, galactose and the mercuric ion.     

PURIFICATION AND PROPERTIES OF HUMAN BRAIN α-L-FUCOSIDASE

Human brain α-L-fucosidase has been extracted and the soluble portion has been purified 9388-fold with 25% yield by a two-step affinity chromatographic procedure utilizing agarose-epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all seven isoelectric forms of the enzyme were purified. Trace amounts of eight glycosidases, with hexosaminidase being the largest contaminant (1% by activity) were found in the purified α-L-fucosidase preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a single subunit of molecular weight 51,000 ± 2500. The purified enzyme has a pH optimum of 4.7 with a suggested second optimum of 6.6. The apparent Michaelis constant and maximal velocity of the purified enzyme with respect to the p-nitrophenyl substrate are 0.44 mM and 10.7 μmol/min/mg protein, respectively. Ag²⁺ and Hg²⁺ completely inactivated the enzyme at concentrations of 0.1-0.3 mM. Antibodies made previously against purified human liver α-L-fucosidase cross-reacted with the purified brain α-L-fucosidase and gave a single precipitin line coincident with that from purified liver α-L-fucosidase. From all our studies it appears that at least the soluble portion of brain α-L-fucosidase is identical to human liver α-L-fucosidase.     

UPTAKE OF α-METHYL-D-GLUCOSIDE BY SYNAPTOSOMES FROM RAT BRAIN

Influx of α-methylglucoside into synaptosomes prepared by differential and Ficoll density gradient centrifugation was studied to determine whether this sugar could be used as a model for glucose transport in nerve endings. The rate of uptake of a-methylglucoside was linear over a wide range of substrate concentrations. Influx was only slightly inhibited (12–15%) in the presence of glucose, 2-deoxyglncose, phloretin and 2,4-dinitrophenol and was unaffected by galactose or phlorizin. Conver- sion of a-methylglucoside to phosphorylated intermediates in synaptosomes was negligible. The data are consislent with the influx of a-methylglucoside being primarily a diffusion process or being mediated by a system with an extremely high K_m. However, it is possible that a small portion of the sugar may be transported by the low affinity glucose transport system. The results indicate that a-methylgluco- side is not a good model for glucose transport in synaptosomes as it is in other tissues.     

A SUBCELLULAR POOL OF HYPO-OSMOTICALLY RESISTANT PARTICLES CONTAINING THYROTROPIN RELEASING HORMONE, α-MELANOCYTE STIMULATING HORMONE, AND LUTEINIZING HORMONE RELEASING HORMONE IN THE RAT HYPOTHALAMUS

Abstract— A 10% homogenate of male rat hypothalami, prepared in 0.32M-sucrose-10μM-CaCl₂, was diluted either with one volume of 0.32M-sucrose-10μM-CaCl₂ (iso-osmotic) or with 10μM-CaCl₂ (hypo-osmotic). A 900 g supernatant fluid fraction (0.9 K-S) was prepared from the diluted homogenates and fractionated on continuous sucrose density gradients under non-equilibrium and equilibrium conditions. In the iso-osmotic 0.9 K-S thyrotropin releasing hormone (TRH), α-melanocyte stimulating hormone (α-MSH), and luteinizing hormone releasing hormone (LHRH) were each found to be sequestered in two populations of particles which were different in size but similar in density. In the hypo-osmotic 0.9 K-S, TRH, α-MSH, and LHRH were each found to be sequestered in a single population of particles. In their sedimentation properties (as judged by differential, non-equilibrium, and equilibrium density centrifugations), the hypo-osmotically resistant particles and the small particles present in the iso-osmotic 0.9 K-S were identical. However, in their peptide content, the two sets of particles differed from each other. If the total quantity of particle-bound peptides recovered after gradient centrifugation of the iso-osmotic 0.9 K-S is taken as 100%, one finds that the amount of TRH, α-MSH. and LHRH recovered in the small particles is 39%, 50%, and 39%. respectively, whereas the amount of TRH, a-MSH, and LHRH recovered in the hypo-osmotically resistant particles is 42%, 68%, and 67%, respectively. This increase in the quantity of peptides sequestered in the small particles occurred concomitantly with the disappearance of peptides from the large synaptosome-like particles. It is estimated that within the large synaptosome-like particles 7% of the TRH, 35% of the a-MSH, and 45% of the LHRH are associated with hypo-osmotically resistant particles. Ultrastructural analysis of purified hypo-osmotically resistant particles containing TRH. α-MSH. or LHRH revealed a predominance of membrane-bounded packets of electron-dense material.     

ADRENERGIC α- AND β-RECEPTORS EXPRESSED BY THE SAME CELL TYPE IN PRIMARY CULTURE OF PERINATAL MOUSE BRAIN

Abstract— The β-adrenergic agonist, isoproterenol and the α- and β-adrenergic agonist. NA. raise the intracellular concentration of cyclic AMP in cultures of dissociated perinatal mouse brain. This rise is prevented by a β- but not by an α-adrenergic antagonist. The maximal level of cyclic AMP reached in the presence of isoproterenol is markedly higher than that found after exposure to NA. However, if NA is used along with an α-adrenergic antagonist, cyclic AMP levels as high as those after isoproterenol are measured. Agonists with α-adrenergic activity including NA decrease the response to isoproterenol. The decrease is blocked by α-adrenergic antagonists. From this and additional evidence it is concluded: (1) The increase in the level of cyclic AMP caused by β-adrenergic agonists is due to β-receptor-mediated stimulation of adenylate cyclase; (2) the inhibition of this effect by α-adrenergic agonists is mediated by adrenergic α-receptors; (3) the α- and β-adrenergic receptors are likely to be located on the same cells, probably the most abundant putative glial precursor cells. The simultaneous stimulation of α- and β-adrenergic receptors on the same cell may be of significance in the regulation of the response to NA.     

α-(γ-AMINOBUTYRYL)-LYSINE IN MAMMALIAN BRAIN: ITS IDENTIFICATION AND DISTRIBUTION

Abstract— α-(γ-Aminobutyryi)-lysine was identified in rabbit brain. This compound was detected exclusively in the brain of mammals, but not in other tissues. It is not concentrated in any particular region of the rabbit brain.     

NEURONAL PIGMENTS: SPECTROSCOPIC CHARACTERIZATION OF HUMAN BRAIN MELANIN

Abstract— A method has been established for the characterization of synthetic melanins obtained from dopamine, norepinephrine, and structurally related compounds which permits the identification of the precursor substrate of the melanin. This is done by interpreting the absorption spectra and the first derivative curves of these melanins solubilized in sodium borohydride. Using this method, the melanin pigment obtained from the substantia nigra of human brain has been shown to be very similar to dopamine-melanin and not DOPA- or norepinephrine-melanins.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

ISOLATION AND IDENTIFICATION OF α-ASPARTYL-SERINE, ALANYLPHENYLALANINE AND ISOLEUCYLLEUCINE FROM CALF BRAIN STEM

Abstract— A reproducible extraction and fractionation scheme for the isolation of peptides from nervous tissue is described. The low molecular weight fraction, obtained by this procedure from calf brain stem tissue, was further investigated. The presence of N -acetyl-aspartic acid, N -acetyl-α-aspartylglutamic acid and pyrrolidone-2-carboxylic acid was confirmed. The occurrence of minor quantities of glutamylpeptides, identical with or related to known γ-glutamyldipeptides and glutathione, was indicated.
Three hitherto unknown dipeptides α-aspartylserine, alanylphenylalanine and isoleucylleucine were identified. Mass spectrometry was used in the final structure analysis.     

THE MECHANISM OF α-ADRENERGIC AGONIST ACTION IN LIVER

1. Although the mechanism of action of a-adrenergic agonists in liver tissue is somewhat complex, a number of experimental approaches can be usefully employed to identify the molecular details of the events that occur. 2. Receptors specific for α₁-adrenergic agonists located on the plasma membranes of rat liver cells have been partially characterized using pharmacological agents, affinity labels and monoclonal antibodies. Much of this work has employed isolated plasma membrane fractions and does not take account of tissue-related factors which may now be studied in the intact perfused rat liver, following the development of an appropriate assay system. 3. Because a redistribution of cellular Ca²⁺ is central to the mechanism of action of a-adrenergic agonists in liver, it is important to first gain an understanding of basic cellular Ca²⁺ regulation. Knowledge about the compartmentation of cellular calcium and about Ca²⁺-translocation systems located in the mitochondria, plasma membrane and endoplasmic reticulum is now quite extensive. However, the role of mitochondria in the regulation of intracellular Ca²⁺ is still unclear; it now appears that the mitochondrial calcium content is much less than considered previously. This may have important implications for such a regulatory role. 4. The sequence of Ca²⁺ movements that may occur when a-adrenergic agonists interact with liver have been identified and are as follows: (a) Ca²⁺ is mobilized from an intracellular pool(s) (mitochondria plus endoplasmic reticulum and/or plasma membranes). (b) This elevates the cytoplasmic free Ca²⁺ concentration and leads to an efflux of the ion from the cell. (c) At this time, Ca²⁺-sensitive metabolic events in the cytoplasm are activated and an increase in Ca²⁺-cycling occurs across the plasma membrane. (d) Immediately after the hormone is withdrawn, there is a net influx of Ca²⁺ into the cell, and the intracellular Ca²⁺ pools and transmembrane fluxes are restored to the pre-induced states. In this model, Ca²⁺ movements across the plasma membrane play a key role in regulating the cytoplasmic Ca²⁺ concentration. 5. In the perfused rat liver it has been possible to define in quite precise terms the amounts and rates of Ca²⁺ mobilized in each of these stages. 6. Although several proposals for ‘second messengers’ to link the hormone-receptor interaction with initial Ca²⁺ mobilization have been made, at this time only polyphosphoinositide turnover appears to be a suitable candidate.     

THE EFFECTS OF TETRAPHENYLBORON ON THE ISOLATION OF NEURONAL AND GLIAL CELLS FROM GUINEA-PIG BRAIN

Abstract— The addition of 20 mM-tetraphenylboron to the tissue-softening medium of a neuronal and glial cell isolation procedure results in a 2-fold and 5-fold increase in the yield of neuronal and glial-enriched material respectively. The purity of the isolated fractions appears unaltered. While tetraphenylboron strongly inhibits protein synthesis, carbonic anhydrase activity and structural integrity appear unaffected by tetraphenylboron. The potential use of this compound in cell isolations is discussed.     

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN GLYCOSAMINOGLYCANS, LIPIDS AND LYSOSOMAL ENZYMES IN MUCOPOLYSACCHARIDOSIS TYPE III B (α-N-ACETYLGLUCOSAMINIDASE DEFICIENCY)

Glycosaminoglycans, lipids and lysosomal enzymes were measured in brain, liver and spleen of a patient with mucopolysaccharidosis Type III B (α-N-Acetylglucosaminidase deficiency). The glycosaminoglycan content of the brain gray and white matter, leptomeninges, spleen and liver of the patient was 4, 3, 10, and 100 times greater than that of the respective tissues of normal controls. Partially degraded heparan sulfate, the concentration of which increased 17 times in the brain, accounted for the increased glycosaminoglycan content of all tissues. The concentration of the gangliosides G_M2, G_M3 and G_D3 was markedly increased in the gray matter, and to a smaller degree in the white matter. Ceramide dihexoside was also increased in the gray matter of the patient with MPS III B. The activity of α-N-Acetylglucosaminidase was absent from the brain and the liver and greatly diminished in the spleen. β-Glucuronidase. β-glucosaminidase and α-l -iduronidase were more active than normally and the activity of α-galactosidase and β-galactosidase was markedly reduced.     

ENZYMES OF THE γ-GLUTAMYL CYCLE IN THE CHOROID PLEXUS AND BRAIN

—The presence of enzymes of the γ-glutamyl cycle in the bovine and rabbit brain and choroid plexus is described. The activities of γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase and γ-glutamyl-cysteine synthetase in the choroid plexus were found to be higher than in the brain. The activity of γ-glutamyl transpeptidase in the choroid plexus was many times higher than the activity of the other enzymes. Brain and choroid plexus γ-glutamyl transpeptidase were activated by Na⁺ and K⁺. Both brain and choroid plexus showed only a very limited capacity to metabolize [¹⁴C]5-oxoproline to ¹⁴CO₂.     

CHANGES IN TITER OF THE BRAIN HORMONE DURING DEVELOPMENT OF THE SILKWORM, BOMBYX MORI

Changes in titer of brain hormone (BH) extracted from the brain, head, and thorax and abdomen during the development of Bombyx mori from the 4th larval molting up to 6 days after adult emergence were investigated. The hypothesis is presented that high secretory activity of the brain is not reflected in a corresponding change in its BH titer, while low secretory activity brings about an accumulation of BH in the brain. An interesting finding is that extremely high titers of BH were found in the thorax and abdomen, from 4 days after pupation to adult emergence, of females only. This sexual difference was, however, not found in all of the races examined.     

MECHANISM OF ACTION OF β-N-OXALYL-L-α, β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN

The source of ammonia in the brain tissue of young rats treated with β-N-oxalyl-l -α, β-diaminopropionic acid (ODAP) has been studied. ODAP administration to 12-day-old rats causes a significant increase in the levels of adenylic acid deaminase in the brain. Glutaminase activity also shows an increase under these conditions. An increase in the levels of acid protease and transglutaminase is also observed in the brain of ODAP-treated animals. Glutamate dehydrogenase activity is decreased slightly. Glutamine synthetase enzyme is not affected. Aspartate-α-ketoglutarate transaminase and aspartate-pyruvate transaminase activities are enhanced in the brain tissue of ODAP-treated rats. It is held that protein degradation, especially the cleavage of free and protein-bound amide bonds, may be responsible for excess ammonia liberation in the brain of ODAP-treated young rats.