In Vivo Methylation of an Arginine in Chicken Myelin Basic Protein

Abstract: The amino acid sequence around the sole methylarginine residue in chicken myelin basic protein was determined and was found to be similar to that previously reported for mammalian myelin basic protein. The ratio N ^G, N '^G-dimethylarginine: N ^G-monomethylarginine:arginine was approximately 1.3:0.9:1.0. No N ^G, N ^G-dimethylarginine was detected in the protein. The in vivo incorporation of methyl groups from [methyl-³H]methionine into methylarginines in myelin was found to occur readily in 2-day-old chickens. Radioactively labelled N ^G, N '^G-dimeth-ylarginine and N ^G-monomethylarginine in myelin were derived solely from myelin basic protein. Radioactivity was also incorporated into N ^G, N ^G-dimeth-ylarginine, although this was not derived from myelin basic protein. As N ^G-monomethylarginine was easily separated from the dimethylarginines, and as it was derived from myelin basic protein, it may be a good marker for myelin basic protein turnover in vivo. A time course study of the incorporation showed that radioactivity was incorporated into N ^G-monomethylarginine up to 6 h after injection, and decayed slowly, with an apparent half-life of about 40 days.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

Characterization of Neuronal Amino Acid Transporters: Uptake of Nitric Oxide Synthase Inhibitors and Implication for Their Biological Effects

Abstract: In the present study we investigated uptake of the nitric oxide (NO) synthase inhibitors N ^G-methyl- l -arginine and N ^G-nitro- l -arginine by the mouse neuroblastoma × rat glioma hybrid cell line NG108-15. Uptake of N ^G-methyl- l -arginine was characterized by biphasic kinetics ( K _m1 = 8 µmol/L, V _max1 = 0.09 nmol × mg⁻¹× min⁻¹; K _m2 = 229 µmol/L, V _max2 = 2.9 nmol × mg⁻¹× min⁻¹) and was inhibited by basic but not by neutral amino acids. Uptake of N ^G-nitro- l -arginine followed Michaelis-Menten kinetics ( K _m = 265 µmol/L, V _max = 12.8 ± 0.86 nmol × mg⁻¹× min⁻¹) and was selectively inhibited by aromatic and branched chain amino acids. Further characterization of the transport systems revealed that uptake of N ^G-methyl- l -arginine is mediated by system y⁺, whereas systems L and T account for the transport of N ^G-nitro- l -arginine. In agreement with these data on uptake of the inhibitors, l -lysine and l -ornithine antagonized the inhibitory effects of N ^G-methyl- l -arginine on bradykinin-induced intracellular cyclic GMP accumulation, whereas l -tryptophan, l -phenylalanine, and l -leucine interfered with the effects of N ^G-nitro- l -arginine. These data suggest that rates of uptake are limiting for the biological effects of NO synthase inhibitors.     

IN VIVO METHYLATION AND TURNOVER OF RAT BRAIN HISTONES

Abstract— The turnover of the different histone components from brain nuclei was studied after the administration of l -[³H]lysine and l -[¹⁴C-methyl]methionine to newborn rats. The radioactivities of the different histone subfractions as well as other proteins were determined over a 280-day period. Biphasic type decay curves (³H and ¹⁴C) were obtained for total brain histones and all the subfractions. From 6 to 40 days of age the half life of total brain histones was 19 days. After reaching brain maturity the half life was 132 days. The lysine rich histone (F₁) was found to turnover the fastest of all the histones, having half lives of 13 and 112 days, respectively. The decay curve for the slightly lysine rich histones (F_2a2, F_2b) gave half lives of 25 days up to 40 days of age and 189 days after reaching brain maturity. The arginine rich histones (F_2a1, F₃) gave a half life of 32 days up to 40 days of age, while no turnover was observed after maturity. The turnover rates of the methyl groups and/or methionyl residues did not vary significantly from the turnover rates of the lysyl residues in the F₂ and F₃ histones. The lysine-rich histones did not contain significant amounts of methionyl residues or methyl groups.
Amino acid analysis of the brain histones revealed that about 3·6 per cent of the lysyl residues in the slightly lysine rich histones were methylated, mainly as ε-N-dimethyllysine. About 13 per cent of the lysyl residues in the arginine rich histones were methylated, mainly as ε-N-monomethyllysine and ε-N-dimethyllysine.     

METABOLISM OF HIGHLY BASIC PROTEINS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

Abstract— (1) The encephalitogenic basic protein obtained from adult rat brain by treatment with 0·03 N-HCl was demonstrable in the brain on the 10th day after birth. It showed a marked increase in quantity during the phase of active myelination.
(2) The proteins extracted under similar conditions from 5-day old rat brain contained several highly basic proteins other than the encephalitogenic basic protein. These basic proteins, which were electrophoretically similar to highly basic proteins extracted similarly from adult rat liver, are histones.
(3) For metabolic studies the entire group of highly basic proteins in the acid extract was obtained after one-step adsorption of other proteins on DEAE-cellulose equilibrated at pH 9·8
(4) After injection of [¹⁴C]lysine the fractions containing highly basic proteins, water soluble non-basic proteins and other tissue proteins of the brain showed higher relative specific radioactivities during the period 1–10 days after birth than during later stages of postnatal development. The fraction containing proteolipid protein, another myelin protein, showed a low relative specific radioactivity throughout the whole period of postnatal development. The relative specific radioactivity of proteolipid protein was somewhat higher in young than in adult rat brain.     

Characterization of l-Arginine and Aminoguanidine Uptake into Isolated Rat Choroid Plexus: Differences in Uptake Mechanisms and Inhibition by Nitric Oxide Synthase Inhibitors

Abstract: Recent reports suggest that nitric oxide (NO) may contribute to several neurodegenerative diseases, e.g., focal cerebral ischemia, N -methyl- d -aspartate-mediated neurotoxicity, and experimental autoimmune encephalomyelitis. Accordingly, an understanding of the CNS transport processes of NO synthase (NOS) inhibitors has important therapeutic implications. The objective of the present study was to characterize the in vitro transport processes governing the uptake of l -[¹⁴C]arginine and the NOS inhibitor [¹⁴C]aminoguanidine in rat choroid plexus tissue. Consistent with previous reports, the uptake of l -[¹⁴C]arginine was mediated by both saturable and nonsaturable processes and was inhibited by the NOS inhibitors N ^G-methyl- l -arginine, N ^G-amino- l -arginine, and N ⁵-imidoethyl- l -ornithine. l -[¹⁴C]Arginine uptake was not inhibited by aminoguanidine or N ^G-nitro- l -arginine. Because aminoguanidine is an organic cation that bears some structural similarity to l -arginine, aminoguanidine might be transported by either an organic cation transporter or by the basic amino acid transporter governing arginine uptake. However, there was no evidence of a saturable uptake process for [¹⁴C]aminoguanidine in isolated rat choroid plexus, in contrast to that observed for l -[¹⁴C]arginine.     

Kinetics, Pharmacology, and Autoradiographic Distribution of l-[3H]Nitroarginine Binding Sites in Rat Cerebellum

Abstract: The kinetics and pharmacology of N ^G-nitro- l -[2,3,4,5-³H]arginine ( l -[³H]NOARG) binding to rat cerebellum were investigated using in vitro radioligand binding. Specific l -[³H]NOARG binding in cerebellum was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. Specific binding was Ca²⁺ dependent and sensitive to pH (with an optimum of 5.5–7.0). Added calmodulin (1.5–40 µg/ml) had no influence on specific l -[³H]NOARG binding. However, the calmodulin antagonists W-5, W-13, and calmidazolium inhibited l -[³H]NOARG binding with IC₅₀ values in the micromolar range, and calmodulin (10 µg/ml) competitively reversed this inhibition. Nitric oxide synthase (NOS) inhibitors ( N ^G-nitro- l -arginine methyl ester and N ^G-monomethyl- l -arginine acetate) and l -arginine displaced l -[³H]NOARG binding with IC₅₀ values in the nanomolar range, whereas d -arginine and basic amino acids ( l -lysine and l -histidine) displaced l -[³H]NOARG binding with IC₅₀ values in the millimolar range. A comparison of the NOS functional assay with l -[³H]NOARG binding in rat cerebellum showed similar profiles of Ca²⁺ dependency and inhibitory kinetics. Quantitative autoradiographic distribution of l -[³H]NOARG binding sites was significantly higher in the molecular layer than in the granular layer of cerebellum. These studies confirm the potential use of l -[³H]NOARG binding to study the regional distribution and functional properties of NOS.     

PROTEIN METHYLATION IN RAT BRAIN IN VITRO

Abstract— Protein-methylation activity in various organs of the rat was studied with S-adenosyl-L-[methyl-¹⁴C]methionine ([methyl-¹⁴C](SAM) as methyl donor. Activity of the enzyme was highest in brain and lowest in liver. Histones comprised approximately 20 per cent of the total radioactivity incorporated, and lysine-rich histone was the most active. Analysis of amino acids of the methylated proteins of rat brain showed arginine to be the amino acid most extensively methylated, but some methylation occurred in lysine residues. An additional [methyl-¹⁴C]-labelled amino acid was found near histidine on the amino acid column chromatogram.     

Molecular genetic characterization of new bovine kappa-casein alleles CSN3F and CSN3G and genotyping by PCR-RFLP

In order to characterize the two new kappa-casein variants F and G (CSN3^F and CSN3^G) recently detected in Ayrshire and Pinzgauer cattle, exon IV of CSN3 from heterozygous animals was amplified by polymerase chain reaction (PCR), cloned and sequenced. The sequencing data revealed single point mutations at nucleotide positions 10530 (G→A) for CSN3^F and 10790 (C→T) for CSN3^G, corresponding to amino acid exchanges in positions 10 (Arg→His) and 97 (Arg→Cys) respectively. These mutations alter recognition sites for the restriction enzymes HhaI and MaeII , which were subsequently used to confirm these polymorphisms in cattle carrying CSN3^F or CSN3^G. A PCR-restriction fragment length polymorphism (RFLP) genotyping procedure for all currently known CSN3 alleles (CSN3^A, CSN3^B, CSN3^C, CSNJ^E, CSN^F, CSN3^G) was developed.     

Isolation and characterization of a microbial Arg/Lys carboxypeptidase, carboxypeptidase F

Carboxypeptidase F was isolated from a fungal strain F-33 and characterized. The enzyme has the ability to release arginine and lysine from the carboxy terminus of peptides, and showed high specific activity against arginine (140 units mg^-1 protein). Optimal temperature and pH for the enzyme reaction were 55°C and pH 8.5, respectively. The enzyme possessed a high thermal stability. Native molecular weight was estimated to be approximately 450000. Enzymatic activity was inhibited by Co²⁺, Cd²⁺, chelating agents and thiol inhibitors.