Proteolytic measurement of mitochondrial aspartate aminotransferase in human serum

Watazu, Yoshifumi; Okabe, Hiroaki; Sugiuchi, Hiroyuki; Uji, Yukitaka; Shirahase, Yasushi; Kaneda, Nobuaki

doi:10.1016/0009-9120(90)80023-c

Cited by 6 publications

(2 citation statements)

References 10 publications

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“…Catalytic activity of mitochondrial aspartate aminotransferase was measured as a marker for the mitochondrial fraction by the malate dehydrogenase-UV method with proteinase-K as described previously (16). Each fraction was separated by 12% SDS-polyacrylamide gel electrophoresis gel and transferred electrophoretically to a polyvinylidene difluoride membrane.…”

Section: Analyses For Subcellular Localization and N-glycosylation Bymentioning

confidence: 99%

Human Mitochondrial Carbonic Anhydrase VB

Fujikawa-Adachi

Nishimori²,

Taguchi

et al. 1999

Journal of Biological Chemistry

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A cDNA clone for a novel carbonic anhydrase (CA) isozyme was isolated from human pancreas and salivary glands. The cDNA sequence of 1182 base pairs encoded a 317-amino acid protein with a predicted mass of 36.4 kDa. The highest similarity of this cDNA and the deduced amino acid sequence is to human CA V (mitochondrial CA), hereafter referred to as CA VA. Recombinant protein expressed in COS-7 cells transfected with this cDNA clone was enriched in a mitochondrial fraction. Confocal fluorescence microscopy showed cytoplasmic granular signals in COS-7 cells expressing a fusion protein of the novel CA and green fluorescent protein. Several lines of evidence suggest that the cDNA clone presented herein encodes a novel human mitochondrial CA isozyme, designated CA VB. CA VB has a hydrophobic N-terminal mitochondrial signal sequence (33 amino acid residues). Western blot analysis showed a 36-kDa protein precursor and a 32-kDa mature protein for CA VB. Similar to CA VA, CA VB is a "low activity" enzyme with a sensitivity to acetazolamide. The CA VB gene is located on Xp22.1. Northern blot analysis in normal human tissues demonstrated expression of a 1.3-kilobase transcript in heart and skeletal muscle, and reverse transcription-polymerase chain reaction analysis showed expression of CA VB in pancreas, kidney, salivary glands, and spinal cord but not in liver. CA VA mRNA expression was observed only in liver. These findings indicate these are two genetically distinct isoforms of human CA V, designated CA VA and CA VB, which have different patterns of tissue-specific distribution, suggest different physiological roles for the two mitochondrial isozymes.

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Section: Analyses For Subcellular Localization and N-glycosylation Bymentioning

confidence: 99%

Human Mitochondrial Carbonic Anhydrase VB

Fujikawa-Adachi

Nishimori²,

Taguchi

et al. 1999

Journal of Biological Chemistry

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“…One unit of proteinase K corresponds to the amount of enzyme which hydrolyses 1 pmoliliter of tyrosine or Folinactive amino acid peptide released from hemoglobin per minute at 37°C and pH 9.0 (9). The assay systems for serum AST and m-AST measurement (protease 401 or proteinase K) are the same as those used in our previous reports (5)(6)(7). Other reagent grade chemicals were obtained from Nakarai Pure Chemical Company (Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase

Watazu

Uji

Okabe

et al. 1993

Clinical Laboratory Analysis

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We studied a new proteinase K assay method for human serum mitochondrial aspartate aminotransferase. We found that proteinase K showed no inactivation of human mitochondrial aspartate aminotransferase isoenzyme and complete inactivation of cytosolic aspartate aminotransferase. Previous studies have shown that selective proteolytic measurement for mitochondrial aspartate aminotransferase in serum using the protease 401 cleaved peptide bond at Leu 20 from the amino-terminal bond shows complete inactivation of cytosolic aspartate aminotransferase and slight inactivation of mitochondrial aspartate aminotransferase isoenzyme, depending on protease concentration. In this investigation, we found that the proteinase K method does not depend on protease concentration. The proteinase K enzyme inactivation of cytosolic aspartate aminotransferase is caused by the cleavage of the peptide bond at Ileu 21 from the aminoterminal bond. In studies with various animal cytosolic aspartate aminotransferase isoenzymes, proteinase K almost completely inactivated cytosolic aspartate aminotransferase. Precision and correlation using proteinase K for measurement of serum mitochondrial aspartate aminotransferase in human showed a good coefficient of variation (within-run < 4.45%) and a coefficient of correlation of r = 0.985 (N = 125).

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Aspartate aminotransferase-linked immunoglobulin complexes in serum of a patient with primary biliary cirrhosis

et al. 1994

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A 51-year-old woman who had been treated for primary biliary cirrhosis (PBC) was admitted to our hospital for evaluation of unexplained, isolated, persistently increased aspartate aminotransferase (AST) activity. Results of laboratory tests on admission showed: AST 171 KU, alanine aminotransferase 28 KU, and anti-mitochondrial titer 1/1280. Results of hepatitis B surface antigen (HBs Ag) and hepatitis C virus antibody (HCV Ab; C100-3) assays were negative. Histology of a liver biopsy specimen was compatible with a diagnosis of PBC (stage III of Scheuer's classification). The molecular size of serum AST was estimated to be more than 500,000 by high-performance size-exclusion liquid chromatography. Electrophoretic analysis showed an abnormal band of AST between supernatant AST (sAST) and mitochondrial AST (mAST), which band was characteristic of AST-immunoglobulin complexes (AST-Ig). Ouchterlony double-diffusion and immunoprecipitation tests identified the immunoglobulin component as IgM. The presence of AST-Ig appeared to be responsible for the elevated serum AST.

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Proteolytic measurement of mitochondrial aspartate aminotransferase in human serum

Cited by 6 publications

References 10 publications

Human Mitochondrial Carbonic Anhydrase VB

Human Mitochondrial Carbonic Anhydrase VB

Proteinase K inactivation of cytosolic aspartate aminotransferase isoenzyme for measurement of human serum mitochondrial aspartate aminotransferase

Aspartate aminotransferase-linked immunoglobulin complexes in serum of a patient with primary biliary cirrhosis

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