Renal synthesis of atriopeptin-like protein in physiology and pathophysiology

1991 ◽  
Vol 260 (4) ◽  
pp. F602-F607 ◽  
Author(s):  
J. E. Greenwald ◽  
P. Needleman ◽  
M. R. Wilkins ◽  
G. F. Schreiner
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Rat Kidney ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Frozen Sections ◽  
Puromycin Aminonucleoside ◽  
Adult Rat ◽  
Major Form ◽  
Anatomic Localization

Atriopeptin is synthesized in mammalian atria as a 126-amino acid (14 kDa) prohormone, but it is secreted and circulates as a 28-amino acid (2.5 kDa) peptide. We have demonstrated the synthesis and secretion of an atriopeptin-like peptide in neonatal and adult rat kidney cell cultures. In this study, we evaluated the site of renal synthesis of this protein and its expression in normal rats and rats made nephrotic with puromycin aminonucleoside. The major form of atriopeptin in normal kidneys comigrated with an apparent molecular mass of 2.5 kDa assessed by gel filtration chromatography. However, the major form of this atriopeptin-like protein in nephrotic kidneys was determined to have an apparent molecular mass similar to the heart prohormone. No atriopeptin prohormone was detected in the plasma of nephrotic rats. Localization of this renal atriopeptin-like protein was accomplished by immunocytochemistry of rat kidney frozen sections. Using an antibody generated against either the COOH-terminal or NH3-terminal region of the cardiac atriopeptin prohormone, we detected specific immunostaining in the distal cortical nephron of the nephrotic kidney. This is the first report of the anatomic localization of a renal atriopeptin-like protein and its upregulation in nephrosis.

scholarly journals Purification and partial structural and kinetic characterization of tyrosine aminotransferase from epimastigotes of Trypanosoma cruzi

1993 ◽  
Vol 292 (3) ◽  
pp. 901-906 ◽  
Author(s):  
M Montemartini ◽  
J A Santomé ◽  
J J Cazzulo ◽  
C Nowicki
Keyword(s):  
Amino Acid ◽  
Trypanosoma Cruzi ◽  
Molecular Mass ◽  
Human Liver ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Tyrosine Aminotransferase ◽  
Apparent Molecular Mass ◽  
Native Enzyme

Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

Physicochemical properties of a novel α-L-arabinofuranosidase fromRhizomucor pusillusHHT-1

2001 ◽  
Vol 47 (8) ◽  
pp. 767-772 ◽  
Author(s):  
A KM Shofiqur Rahman ◽  
Shinya Kawamura ◽  
Masahiro Hatsu ◽  
M M Hoq ◽  
Kazuhiro Takamizawa
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Rhizomucor Pusillus ◽  
Terminal Amino ◽  
Metal Cofactors

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

scholarly journals Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

scholarly journals Studies on the subunits of human myeloperoxidase

1984 ◽  
Vol 222 (3) ◽  
pp. 701-709 ◽  
Author(s):  
R L Olsen ◽  
C Little
Keyword(s):  
Heat Treatment ◽  
Amino Acid ◽  
Molecular Mass ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Additional Species ◽  
Main Protein

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.

scholarly journals Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

2000 ◽  
Vol 345 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Bruno ANTONSSON ◽  
Sylvie MONTESSUIT ◽  
Sandra LAUPER ◽  
Robert ESKES ◽  
Jean-Claude MARTINOU
Keyword(s):  
Cytochrome C ◽  
Molecular Mass ◽  
Lipid Membranes ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Cytochrome C Release ◽  
Octyl Glucoside ◽  
Apoptotic Activity

Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.

scholarly journals Cleavage state of γENaC in mouse and rat kidney

2021 ◽  
Author(s):  
Gustavo Frindt ◽  
Shujie Shi ◽  
Thomas R Kleyman ◽  
Lawrence G Palmer
Keyword(s):  
Molecular Mass ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Mouse Kidney ◽  
Apparent Molecular Mass ◽  
Na Channel ◽  
Extracellular Proteases ◽  
Salt Diet ◽  
Heterologous System ◽  
Membrane Bound

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the g subunit. Here we investigate the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in FRT cells by co-expressing the apical-membrane bound protease CAP1 (prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest C-terminal fragment of gENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in FRT cells. Similar cleaved peptides were observed in total and cell surface fractions of rat kidney. This suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using non-reducing gels, in which the N- and C-terminal fragments of gENaC are linked by a disulfide bond. Under these conditions the major cleaved form in rat kidney had an apparent molecular mass of 56 kDa, ~4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant gENaC species in the apical membrane of rat and mouse kidney on a low-Na diet is the twice-cleaved, presumably activated form.

scholarly journals N-acetyl-d-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1

1996 ◽  
Vol 317 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Miguel A. FERRERO ◽  
Angel REGLERO ◽  
Manuel FERNANDEZ-LOPEZ ◽  
Roberto ORDAS ◽  
Leandro B. RODRIGUEZ-APARICIO
Keyword(s):  
Escherichia Coli ◽  
Sialic Acid ◽  
Amino Acid ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Neuraminic Acid ◽  
Acid Biosynthesis ◽  
Colominic Acid ◽  
Apparent Homogeneity ◽  
Escherichia Coli K1

Colominic acid is a capsular homopolymer from Escherichia coli K1 composed of α(2-8)-linked N-acetyl-d-neuraminic acid (NeuAc) residues. Recently, we have described that NeuAc synthesis in this bacterium occurs through the action of NeuAc lyase (EC 4.1.3.3) [Rodríguez-Aparicio, Ferrero and Reglero (1995) Biochem. J. 308, 501–505]. In the present work we analysed and characterized this enzyme. E. coli K1 NeuAc lyase is detected from the early logarithmic phase of growth, is induced by NeuAc and is not repressed by glucose. The enzyme was purified to apparent homogeneity (312-fold) using two types of hydrophobic chromatographies (butyl-agarose and phenyl-Sepharose CL-4B), gel filtration on Sephacryl S-200, and anion-exchange chromatography on DEAE-FPLC. The pure enzyme, whose amino acid composition and N-terminal amino acid sequence are also established, has a native molecular mass, estimated by gel filtration, of 135±3 kDa, whereas its molecular mass in SDS/PAGE was 33±1 kDa. The enzyme was able to synthesize and cleave NeuAc in a reversible reaction. The maximal rate of catalysis was achieved in 125 mM Tris/HCl buffer, pH 7.8, at 37 °C. Under these conditions, the Km values calculated for N-acetyl-d-mannosamine and pyruvate (condensation direction), and NeuAc (hydrolysis direction) were 7.7, 8.3 and 4.8 mM respectively. NeuAc synthesis by the pure enzyme was activated by Ca2+ and inhibited by Mn2+ and NeuAc, whereas the enzyme cleavage direction was inhibited by Ca2+, Mn2+ and pyruvate. The reaction products, NeuAc and pyruvate, and Ca2+ are able to regulate the direction of this enzyme (synthesis or cleavage of sialic acid) and, accordingly, to modulate colominic acid biosynthesis.

scholarly journals Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

1999 ◽  
Vol 344 (2) ◽  
pp. 331-337 ◽  
Author(s):  
Lakhdar GASMI ◽  
Jared L. CARTWRIGHT ◽  
Alexander G. MCLENNAN
Keyword(s):  
Fusion Protein ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Apparent Molecular Mass ◽  
Protein Glycation ◽  
Human Homologue ◽  
Protein Fragment ◽  
Sds Page

The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5′-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. Km and kcat values with ADP-ribose were 60 μM and 5.5 s-1 respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg2+ or 100-250 μM Mn2+ ions; fluoride was inhibitory, with an IC50 of 20 μM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD+ and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation.

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