N- and C-terminal amino acids of proteolipids from the rat brain and various other organs

N- and C-terminal amino acids of proteolipid proteins from the whole brain and some other organs were investigated. N-terminal amino acids were identified by the dansylation procedure. C-terminal amino acids were determined after the enzymatic hydrolysis with carboxy peptidases A and B with the following dansylation. Phenyl alanine and lysine were identified as C-terminal amino acids of the proteolipids from the whole brain and only lysine--as the C-terminal amino acid of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle. The corresponding N-terminal amino acids of the proteolipids from the whole brain were aspartic acid and glycine and of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle--only aspartic acid. A comparison of the data obtained with the previous ones has shown that in the brain there exist only two types of proteolipids--one characteristic of myelin, another-- of mitochondria, and in other organs--only one characteristic of mitochondria.     

Comparison of turnover rates of proteins of the brain, liver and kidney in mouse in vivo following long term labeling.

Intraperitoneal injection of [14C]tyrosine suspension followed by subcutaneous implantation of a [14C]tyrosine pellet in mice produced a fairly constant specific activity of plasma free tyrosine for 5 days, and for 3-5 days in the tissue free amino acid pool. The specific activity of tyrosine in the tissue (brain, liver, and kidney) free amino acid pool was 75-90% of that in plasma. Incorporation of tyrosine into tissue proteins was followed for 5 days in brain; during this time 33% of tissue proteins were labeled. Incorporation for 68 h in liver and kidney showed labeling of over 70% of the protein of these tissues. These percentages assume a homogeneous tissue free tyrosine pool as the precursor. The rate of incorporation initially was 0.6, 2.8, and 2.0% per h in brain, liver, and kidney protein, respectively. These rates decreased in longer term experiments. The best fit to the incorporation curves was obtained by assuming the following average half-lives for tissue proteins: brain, two compartments, 5.7% with a half-life of 15 h, 94.3% with a half-life of 10 days; liver, a single compartment with a 26-h half-life; kidney, two compartments, 41% with an 18-h half-life, and 59% with a 63-h half-life.     

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain

Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.     

Studies on the catabolism of Ng-methylarginine, Ng, Ng-dimethylarginine and Ng, Ng-dimethylarginine in the rabbit.

1. The routes of elimination of Ng-methylarginine, Ng, Ng-dimethylarginine and Ng, Ng-dimethylarginine were investigated in the rabbit. 2. Analyses showed low plasma concentrations of these amino acids (around 1 nmol/ml) and ratios similar to those found in tissue proteins. The concentrations of these amino acids in extracts of brain, kidney, liver and spleen were similar except that liver had a lower concentration of Ng-methylarginine and Ng, Ng-dimethylarginine. Cerebrospinal fluid contained traces of each amino acid.     

Characterization of the cDNA Coding for Rat Brain Cysteine Sulfinate Decarboxylase

Cysteine sulfinate decarboxylase (CSD) is considered as the rate-limiting enzyme in the biosynthesis of taurine, a possible osmoregulator in brain. Through cloning and sequencing of RT-PCR and RACE-PCR products of rat brain mRNAs, a 2,396-bp cDNA sequence was obtained encoding a protein of 493 amino acids (calculated molecular mass, 55.2 kDa). The corresponding fusion protein showed a substrate specificity similar to that of the endogenous enzyme. The sequence of the encoded protein is identical to that encoded by liver CSD cDNA. Among other characterized amino acid decarboxylases, CSD shows the highest homology (54%) with either isoform of glutamic acid decarboxylase (GAD65 and GAD67). A single mRNA band, approximately 2.5 kb, was detected by northern blot in RNA extracts of brain, liver, and kidney. However, brain and liver CSD cDNA sequences differed in the 5' untranslated region. This indicates two forms of CSD mRNA. Analysis of PCR-amplified products of genomic DNA suggests that the brain form results from the use of a 3' alternative internal splicing site within an exon specifically found in liver CSD mRNA. Through selective RT-PCR the brain form was detected in brain only, whereas the liver form was found in liver and kidney. These results indicate a tissue-specific regulation of CSD genomic expression.     

Metabolism and transport of gamma-carboxyglutamic acid

gamma-Carboxyglutamic acid residues have beeh shown to be present in prothrombin, the other vitamin K-dependent clotting factors, and more recently in bone and kidney proteins. This amino acid is formed by a posttranslational vitamin K-dependent carboxylation of glutamyl residues in polypeptide precursors of these protens. It has now been demonstrated that this amino acid, either in the free or peptide-bound form, is not metabolically degraded by the rat, but is quantitatively excreted in the urine. In nephrectomized rats, the tissue concentration of intravenously administered gamma-carboxyglutamic acid is increased, but there is still no evidence of any oxidative metabolism of this amino acid. These amino acid is transported by kidney slices against a concentration gradient, but does not accumulate in liver, intestinal or brain tissues. Preliminary data suggest that gamma-carboxyglutamic acid may be concentrated by a carrier system different from that utilized by other amino acids.     

Amino- and carboxyl-terminal amino acids of proteolipid proteins

The amino- and carboxyl-terminal amino acids of proteolipids from neural and non-neural sources were investigated. Amino-terminal amino acids were identified and quantitated by the dansyiation procedure. Carboxyl-terminal amino acids were determined after hydrazinolysis or enzymatic hydrolysis with carboxypeptidases. Proteolipid from white matter showed two terminal amino acids, regardless of the method of preparation. The major N-terminal amino acid was glycine and the minor one was glutamic acid or glutamine. The corresponding C-terminal amino acids were phenylalanine and glycine. Preparations of white matter proteolipid, therefore, contained more than one protein or protein chain. Proteolipids from brain mitochondria, heart, liver and kidney were characterized by N-terminal aspartic acid or asparagine and C-terminal lysine residues and they exhibited an amino acid composition which differed from white matter proteolipid. Our results suggest the existence of two classes of proteolipids, a myelin type and a non-myelin type. Synaptic membrane and grey matter proteolipids exhibited characteristics of both classes.     

Brain and Kidney d-Amino Acid Oxidases Are Coded by a Single Gene in the Mouse

Mutant mice (ddY/DAO-) lacking D-amino acid oxidase in the kidney also lacked this enzyme in the brain. Genetic cross experiments showed that the inheritance of the enzyme in the brain was the same as that in the kidney. The deficiency in the enzyme in the brain could not be separated from that in the kidney. The brain and kidney enzymes showed similar substrate specificities. These results suggest that brain and kidney D-amino acid oxidases are coded by the same gene in the mouse.     

Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.     

大鼠肾硫酸基转移酶基因的cDNA克隆及序列分析

在许多激素、神经递质、药物和异生化合物的生物转化中,硫酸酯化反应是一重要的代谢途径［１,２］．这些化合物的硫酸酯化通常导致其生物活性的降低及尿排泄量的增加．硫酸酯化是把３′－磷酸腺苷－５′磷酸硫酸（ＰＡＰＳ）的活性硫酸根转移到某一底物（如Ｒ－ＯＨ）．...     

Acidic amino acid transport in animal cells and tissues

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)     

PROLIDASE ACTIVITY IN BRAIN: COMPARISON WITH OTHER ORGANS

Prolidase (imidodipeptidase, EC 3.4.3.7) activity in brain was assayed spectrophotometrically, and the validity of the assay was confirmed by microchromatographic analysis of the dansyl derivatives of the reaction products. The ratio of the activity of prolidase from brain on Ala-Pro, Gly-Pro, and Pro-Pro was similar to that of the highly purified intestinal mucosal prolidase. The size and polarity of the amino acid side chain (R) of the N-terminal amino acid and whether the terminal amino and carboxy groups are free or blocked are important factors in determining prolidase substrate activity. Using Ala-Pro as substrate, we found prolidase activity in all rat tissues we investigated, with highest activity in kidney and lowest in plasma; 33 mmol of Ala-Pro was hydrolyzed per mg of protein per min by brain tissue, which is 20% of the activity for kidney and 23% of that for intestinal mucosa. The specific activity of prolidase was higher in the sciatic nerve than in brain or spinal cord, and activity was higher in the distal portion.     

Isolation of mouse and human cDNA clones encoding a protein expressed specifically in osteoblasts and brain tissues.

Using the differential hybridization screening method between osteoblastic and fibroblastic cells, a cDNA clone coding for an osteoblast specific protein, named OSF-1, consisting of 168 amino acid residues including a possible 32 amino acid long leader sequence, was isolated from murine osteoblastic cell line MC3T3-E1. The OSF-1 gene was shown by Northern blotting analysis to be expressed in mouse calvarial osteoblast-enriched cells and in mouse brain tissues, but not in thymus, spleen, kidney, liver, lung, testis or heart. The human counterpart was also found in cDNA libraries from human osteosarcoma cell line MG63 and normal brain tissues. DNA sequence analysis revealed four amino acid sequence differences between the mouse and human, of which only one is located in the mature protein. This extremely high sequence conservation suggests that OSF-1 plays a fundamental role in bone and brain functions.     

Molecular cloning and sequence analysis of cDNA coding for rat liver hemoprotein H-450

cDNA clones coding for hemoprotein H-450 were isolated from a rat liver cDNA library using anti-H-450 antibody. The molecular weight calculated from the deduced amino acid sequence comprising 547 amino acid residues was 60,085. The N-terminal sequence and a partial internal amino acid sequence of purified H-450, which were determined chemically, were both found in the amino acid sequence of H-450 deduced from the nucleotide sequence. H-450 mRNA is expressed in liver, kidney, and brain. A homology search of amino acid sequences indicated that H-450 shows no homology with cytochrome P-450, but shows significant homology with bacterial O-acetylserine (thiol)-lyases. However, H-450 has no O-acetylserine (thiol)-lyase activity.