scholarly journals Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies

1991 ◽  
Vol 278 (1) ◽  
pp. 171-177 ◽  
Author(s):  
A J Rivett ◽  
S T Sweeney
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Rat Tissues ◽  
Specific Antibodies ◽  
Multicatalytic Proteinase ◽  
Cell Extracts ◽  
Liver And Kidney ◽  
Lysosomal Proteinase

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

scholarly journals Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

1998 ◽  
Vol 180 (17) ◽  
pp. 4591-4595 ◽  
Author(s):  
Zhongqi He ◽  
John K. Davis ◽  
Jim C. Spain
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Pseudomonas Pseudoalcaligenes ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts ◽  
High Degree

ABSTRACT 2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.

Distribution of the adrenocortical inner zone antigen

1994 ◽  
Vol 141 (3) ◽  
pp. 459-466 ◽  
Author(s):  
M M Ho ◽  
S Barker ◽  
G P Vinson
Keyword(s):  
Molecular Mass ◽  
Adrenal Cortex ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Detection Methods ◽  
Rat Tissues ◽  
Renal Tubules ◽  
Immunoreactive Protein

Abstract Previous studies using a mouse monoclonal antibody (IZAb) have identified inner zone-specific antigens (IZAg 1 and 2) in the rat adrenal cortex. The expression of IZAgl is enhanced by dibutyryl-cyclic AMP or ACTH in vitro and by in vivo ACTH treatment. These studies also suggest that IZAg may be involved in steroidogenesis in rat adrenals. Using sensitive detection methods, the distribution of IZAg in adrenals from other species and in other rat tissues was studied. Pig and rabbit adrenals expressed an IZAb-immunoreactive protein with molecular mass identical with that of IZAg1, but no immunoreactivity was detected in bovine, guinea-pig or mouse adrenals. In the rabbit adrenal, as in the rat, IZAb bound only to zonae fasciculata/reticularis cells. However, the whole adrenal cortex in the pig immunoreacted with IZAb. Although immunofluorescence was observed in human adrenal sections, predominantly in the zona reticularis, no significant immunoreactive protein band was detected by immunoblot analysis. Apart from the zonae fasciculata/reticularis in the adrenal gland, an IZAb-immunoreactive protein with a molecular mass of 26 000 Da, corresponding to IZAg1, was also found in rat hepatocytes, renal tubules, ovary (corpus luteum, interstitial cells, theca interna of mature follicles) and Leydig cells. The observations suggest that IZAg may be involved in the processes of biosynthesis, metabolism and/or excretion of steroid hormones. Journal of Endocrinology (1994) 141, 459–466

scholarly journals Purification and further characterization of peroxisomal trihydroxycoprostanoyl-CoA oxidase from rat liver

1994 ◽  
Vol 304 (1) ◽  
pp. 195-200 ◽  
Author(s):  
P P Van Veldhoven ◽  
P Van Rompuy ◽  
J C T Vanhooren ◽  
G P Mannaerts
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Gel Filtration ◽  
Chromatographic Separations ◽  
Metal Affinity ◽  
Native Molecular Mass ◽  
Native Gel ◽  
Acyl Coa Oxidase ◽  
Subunit Size

The acyl-CoA oxidase, catalysing the peroxisomal desaturation of the CoA-ester of trihydroxycoprostanic acid, a bile acid intermediate, has been purified to homogeneity from rat liver. Its native molecular mass, as determined by gel filtration and native gel electrophoresis, was 120 and 175 kDa respectively, suggesting a homodimeric protein consisting of 68.6 kDa subunits. If isolated in the presence of FAD, the enzyme showed a typical flavoprotein spectrum and contained most likely 2 mol of FAD per mol of enzyme. The cofactor, however, was loosely bound. The enzyme acted exclusively on 2-methyl-branched compounds, including pristanoyl-CoA and 2-methylhexanoyl-CoA if albumin was present. Important parameters to obtain a pure and active enzyme were the following: (1) using chromatographic separations like hydrophobic interaction and metal affinity, which allow the presence of high salt concentrations, conditions which stabilize the oxidase; (2) avoiding dialysis and (NH4)2SO4 precipitation; (3) including, when appropriate, FAD, dithiothreitol and a diol-compound in the solvents; and (4) carefully monitoring the removal of other acyl-CoA oxidases which possess the same native molecular mass and subunit size.

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.

scholarly journals Formation of complexes between 125I-labelled human or bovine somatotropins and binding proteins in vivo in rat liver and kidney

1983 ◽  
Vol 214 (1) ◽  
pp. 121-132 ◽  
Author(s):  
J S Bonifacino ◽  
L P Roguin ◽  
A C Paladini
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Radioactive Material ◽  
Bovine Somatotropin ◽  
Liver Uptake ◽  
Liver And Kidney ◽  
Specific Proteins ◽  
Triton X ◽  
Formation Of Complexes

At 5 min after intravenous injection, both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin were concentrated in rat liver and kidney. When the labelled hormones were administered along with an excess of the corresponding unlabelled hormone, a significant decrease of the uptake was observed in the liver, but not in the kidney. Study of the subcellular distribution of radioiodinated somatotropins in liver revealed that most of the radioactivity was specifically concentrated in the microsomal fraction. In contrast, the kidney fraction that accounted for most of the radioactivity was the 100 000 g supernatant. After solubilization, with 1% (w/v) Triton X-100, of the microsomal fractions obtained from both organs, the radioactive material was analysed by gel filtration on Sepharose CL-6B. By using this approach, it was demonstrated that both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin bind in vivo to proteins present in liver. A small proportion of 125I-labelled human somatotropin was also shown to form complexes with proteins present in kidney. The present results demonstrate that the liver uptake is mainly due to binding of somatotropins to specific proteins, in contrast with the kidney, in which binding to specific sites contributes minimally to the overall uptake.

scholarly journals Problems associated with the assay of arylsulphatases A and B of rat tissues

1973 ◽  
Vol 134 (1) ◽  
pp. 183-190 ◽  
Author(s):  
Mark Worwood ◽  
Kenneth S. Dodgson ◽  
Gary E. R. Hook ◽  
Frederick A. Rose
Keyword(s):  
Rat Liver ◽  
Liver Enzymes ◽  
Gel Filtration ◽  
Precise Determination ◽  
Approximate Determination ◽  
Rat Tissues ◽  
Human Tissues ◽  
Separation And Purification ◽  
The Individual

A method for the separation and purification of rat liver arylsulphatases A and B by gel filtration on Sephadex G-200 is described. The properties of the A enzyme and its molecular weight are similar to those of the corresponding ox liver enzyme. The B enzymes were found to be dissimilar. The method already developed for the assay of the corresponding enzymes from human tissues was shown to be unsuitable for the assay of the enzymes of rat tissues. A method of assay was developed which permits an approximate determination of the individual rat liver enzymes in a mixture of the two, but precise determination requires prior separation of the enzymes by gel-filtration chromatography.

scholarly journals Purification of a protease in red blood cells that degrades oxidatively damaged haemoglobin

1991 ◽  
Vol 277 (3) ◽  
pp. 779-786 ◽  
Author(s):  
J M Fagan ◽  
L Waxman
Keyword(s):  
Red Blood Cells ◽  
Molecular Mass ◽  
Blood Cells ◽  
Gel Filtration ◽  
Cell Types ◽  
Insulin Degrading Enzyme ◽  
Multicatalytic Proteinase ◽  
Proteolytic Pathway ◽  
A Subunit ◽  
Hydrolysis Of

Haemoglobin damaged by exposure of red blood cells to oxidants is rapidly degraded by a proteolytic pathway which does not require ATP [Fagan, Waxman & Goldberg (1986) J. Biol. Chem. 261, 5705-5713]. By fractionating erythrocyte lysates, we have purified two proteases which hydrolyse oxidatively damaged haemoglobin (Ox-Hb). One protease hydrolysed small fluorogenic substrates in addition to Ox-Hb. Its molecular mass was approximately 700 kDa and it consisted of several subunits ranging in size from 22 to 30 kDa. This enzyme may be related to the high-molecular-mass multicatalytic proteinase previously isolated from a variety of tissue and cell types. The other Ox-Hb-degrading activity had an apparent molecular mass of 400 kDa on gel filtration, a subunit size of 110 kDa and an isoelectric point between 4.5 and 5.0. This protease also hydrolysed the small polypeptides insulin and glucagon, as well as other large proteins such as lysozyme. Insulin blocked the degradation of Ox-Hb and Ox-Hb blocked the hydrolysis of insulin by the purified protease. Thiol reagents and metal chelators strongly inhibited the hydrolysis of both Ox-Hb and insulin, whereas inhibitors of serine, aspartic and thiol proteases had little effect. These properties suggest that the Ox-Hb-degrading activity purified from rabbit erythrocytes is the cytosolic insulin-degrading enzyme that is believed to play a role in the metabolism of insulin in several tissues. We propose that this enzyme may also function as a key component in a cytoplasmic degradative pathway responsible for removing proteins damaged by oxidants.

Expression and localization of rat NBC4c in liver and renal uroepithelium

2004 ◽  
Vol 287 (3) ◽  
pp. C781-C789 ◽  
Author(s):  
Natalia Abuladze ◽  
Alexander Pushkin ◽  
Sergei Tatishchev ◽  
Debra Newman ◽  
Pakan Sassani ◽  
...  
Keyword(s):  
Sodium Bicarbonate ◽  
Molecular Mass ◽  
Rat Liver ◽  
Rat Kidney ◽  
Intrahepatic Bile Duct ◽  
Rt Pcr ◽  
Urine Ph ◽  
Intrahepatic Bile Ducts ◽  
Liver And Kidney ◽  
Basolateral Plasma Membrane

Previous studies provided functional evidence for electrogenic Na+-HCO3− cotransport in hepatocytes and in intrahepatic bile duct cholangiocytes. The molecular identity of the transporters mediating electrogenic sodium-bicarbonate cotransport in the liver is currently unknown. Of the known electrogenic Na+-HCO3− cotransporters (NBC1 and NBC4), we previously showed that NBC4 mRNA is highly expressed in the liver. In the present study, we performed RT-PCR, immunoblotting, and immunohistochemistry to characterize the expression pattern of NBC4 in rat liver and kidney. For immunodetection, a polyclonal antibody against rat NBC4 was generated and affinity purified. Of the known human NBC4 variants, only the rat NBC4c ortholog was detected by RT-PCR in rat liver, and the molecular mass of the NBC4c protein was ∼145 kDa. NBC4c protein was expressed in hepatocytes and in the cholangiocytes lining the intrahepatic bile ducts. In hepatocytes, NBC4c was localized to the basolateral plasma membrane, whereas intrahepatic cholangiocytes stained apically. The NBC1 electrogenic sodium cotransporter variants kNBC1 and pNBC1 were not detected by immunoblotting and immunohistochemistry in rat liver. The pattern of localization of NBC4c in the liver suggests that the cotransporter plays a role in mediating Na+-HCO3− cotransport in hepatocytes and intrahepatic cholangiocytes. Unlike the liver, the rat kidney expressed electrogenic sodium-bicarbonate cotransporter proteins kNBC1 and NBC4c. In kidney, NBC4c also had a molecular mass of ∼145 kDa and was immunolocalized to uroepithelial cells lining the renal pelvis, where the cotransporter may play an important role in protecting the renal parenchyma from alterations in urine pH.

Elemental and molecular mass spectrometry for integrated selenosugar speciation in liver and kidney tissues of maternal feeding and supplemented rats

2015 ◽  
Vol 30 (1) ◽  
pp. 267-276 ◽  
Author(s):  
Héctor González-Iglesias ◽  
María Luisa Fernández-Sánchez ◽  
Ying Lu ◽  
Sonia Fernández Menéndez ◽  
Spiros A. Pergantis ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Rat Liver ◽  
Ion Sources ◽  
Selenium Speciation ◽  
Molecular Ion ◽  
Molecular Mass Spectrometry ◽  
Liver And Kidney

The integrated use of elemental and molecular ion sources for enhanced selenium speciation data in rat liver and kidneys, along with its potential for Se-supplement studies are highlighted.

scholarly journals Identification of bile acid-CoA: amino acid N-acyltransferase in rat kidney

1991 ◽  
Vol 280 (3) ◽  
pp. 821-824 ◽  
Author(s):  
J B Kwakye ◽  
M R Johnson ◽  
S Barnes ◽  
W E Grizzle ◽  
R B Diasio
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Molecular Mass ◽  
Rat Liver ◽  
Polyclonal Antibody ◽  
Human Liver ◽  
Acid Metabolism ◽  
Rat Kidney ◽  
Immunoblot Analysis ◽  
Bile Acid Metabolism

A novel location of the bile-acid-conjugating enzyme bile acid-CoA:amino acid N-acyltransferase (BAT) has been discovered in the cytosolic fraction of rat kidney. Both taurine and glycine were utilized as substrates. Formation of bile acid N-acyl amidates was verified by h.p.l.c. by comparison with authentic standards and by specific hydrolysis using cholylglycine hydrolase. Immunoblot analysis using a human liver anti-BAT polyclonal antibody indicated that rat kidney BAT has the same molecular mass as rat liver BAT. These findings suggest that the kidney has a role in bile acid metabolism and physiology.

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