Skip to main content
SpringerLink
Log in

First cloning and characterization of aspartate aminotransferase from river buffalo (Bubalus bubalis)

  • Section Cellular and Molecular Biology
  • Published:
Biologia Aims and scope Submit manuscript

Abstract

Aspartate aminotransferase catalyzes the transfer of an amino group from L-aspartate to α-oxoglutarate. We have purified cytosolic isozyme, to apparent homogeneity, from the heart of river buffalo. In order to clone the enzyme total RNA was isolated and the cDNA encoding complete polypeptide of 413 amino acids was amplified by reverse transcriptase polymerase chain reaction. The cDNA and the deduced amino acid sequence exhibited 98.2% and 99.5% identities, respectively, with that of cow. The cDNA was overexpressed in Escherichia coli and the gene product was obtained in enzymologically active form. The recombinant enzyme was about 40% of the total cell proteins. The purified recombinant enzyme displayed specific activity, Km, temperature and pH stability values similar to that of the native enzyme. Edman degradation and electrospray ionization mass spectra showed that the recombinant enzyme consisted of three species having N-terminal sequences of MAPPSIF, APPSIF and PPSIF, although the second one was the predominant species. Conditions are also described, which in the mass spectral analysis give either the three species corresponding to the apo- or the holo-enzyme; the latter retaining pyridoxal phosphate bound to the protein. To our knowledge this is the first report on sequence determination, cloning and characterization of aspartate aminotransferase from river buffalo.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AST:

aspartate aminotransferase

AST-Bb :

aspartate aminotransferase from Bubalus bubalis

CBB:

coomassie brilliant blue

DDBJ:

DNA Data Bank of Japan

ESI:

electrospray ionization

IPTG:

isopropyl-β-Dthiogalactopyranoside

LC/MS:

liquid chromatography mass spectrometry

NADH:

nicotinamide adenine dinucleotide

PAGE:

polyacrylamide gel electrophoresis

PCR:

polymerase chain reaction

PLP:

pyridoxal phosphate

SDS:

sodium dodecyl sulfate

TIC:

total ion current

TFA:

trifluoroacetic acid

TOF:

time of flight

References

  • Altschul S.F., Gish W., Miller W., Myers E.W. & Lipman D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410.

    PubMed  CAS  Google Scholar 

  • Banks B.E.C., Doonan S., Lawrence A.J. & Vernon C.A. 1968. The molecular weight and other properties of aspartate aminotransferase from pig heart muscle. Eur. J. Biochem. 5: 528–539.

    Article  PubMed  CAS  Google Scholar 

  • Bhat P.N. 1992. Genetics of river buffaloes, pp. 13–58. In: Tulloh N.M. & Holmes J.H.G. (eds), Buffalo Production, Elsevier, Amsterdam.

    Google Scholar 

  • Benson D.A., Karsch-Mizrachi I., Lipman D.J., Ostell J. & Sayers E.W. 2011. GenBank. Nucleic Acids Res. 39: D32–D37.

    Article  PubMed  Google Scholar 

  • Bilal M.Q., Suleman M. & Raziq A. 2006. Buffalo: black gold of Pakistan. Livest. Res. Rural. Develop. 18: 128–135.

    Google Scholar 

  • Frank J.T., Barbara J.W. & Benjamin F.M. 1990. Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures. Plant Physiol. 92: 587–594.

    Article  Google Scholar 

  • Fukumoto Y., Anase S., Nagashima F., Uedas S., Ikegami K.S. & Morino Y. 1991. Structural and functional role of the aminoterminal region of porcine cytosolic aspartate aminotransferase: catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side. J. Biol. Chem. 266: 4187–4193.

    PubMed  CAS  Google Scholar 

  • Han Q., Fang J. & Li J. 2001. Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase. Biochem. J. 360: 617–623.

    Article  PubMed  CAS  Google Scholar 

  • Horio Y., Tanaka T., Taketoshi M., Nagashima F., Tanase S., Morino Y., Wada H. 1988. Rat cytosolic aspartate aminotransferase: molecular cloning of cDNA and expression in Escherichia coli. J. Biochem. 103: 797–804.

    PubMed  CAS  Google Scholar 

  • Ivanov V.I. & Karpeisky M.Y. 1969. Dynamic three-dimensional model for enzymic transamination. Adv. Enzymol. Related Areas Mol. Biol. 32: 21–53.

    CAS  Google Scholar 

  • Johnson R.D., O’Connor M.L. & Kerr R.R. 1995. Extreme serum elevations of aspartate aminotransferase. Am. J. Gastroenterol. 90: 1244–1245.

    PubMed  CAS  Google Scholar 

  • Juretic N., Mattes U., Ziak M., Christen P. & Jaussi R. 1990. Structure of the genes of two homologous intracellularly heterotopic isoenzymes cytosolic and mitochondrial aspartate aminotransferase of chicken. Eur. J. Biochem. 192: 119–126.

    Article  PubMed  CAS  Google Scholar 

  • Kim H.C., Nam C.M., Jee S.H., Han K.H. & Oh D.K. 2004. Normal serum aminotransferase concentration and risk of mortality from liver diseases. prospective coherent study. Brit. Med. J. 328: 983–987.

    CAS  Google Scholar 

  • Kirsch J.F., Eichele G., Ford G.C., Vincent M.G., Jansonius J.N., Gehring H. & Christen P. 1984. Mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. J. Mol. Biol. 174: 497–525.

    Article  PubMed  CAS  Google Scholar 

  • Lacour B., Vassault A., Degos F., Bruneau M., Kreis H. & Bailly M. 1982. In vitro supplementation of pyridoxal phosphate for the optimisation of the determination of the catalytic activity of alanine aminotransferase and aspartate aminotransferase in kidney transplant patients. Clin. Chim. Acta 120: 1–12.

    Article  PubMed  CAS  Google Scholar 

  • Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  PubMed  CAS  Google Scholar 

  • Marino G., Greco A.M., Scardi V. & Zito R. 1966. Purification and general properties of aspartate aminotransferase of ox heart. Biochem. J. 99: 589–594.

    PubMed  CAS  Google Scholar 

  • Marino G., Nitti G., Arnone M.I., Sannia G., Gambacorta A. & De Rosa M. 1988. Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus. J. Biol. Chem. 263: 12305–12309.

    PubMed  CAS  Google Scholar 

  • Martinez-Carrion M., Tiemeier D.C. & Peterson D.L. 1970. Conformational properties of the isoenzymes of aspartate transaminase and the enzyme-substrate complexes. Biochemistry 9: 2574–2582.

    Article  PubMed  CAS  Google Scholar 

  • Mohamed T.M. 2001. Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii. Exp. Appl. Acarol. 25: 231–244.

    Article  PubMed  CAS  Google Scholar 

  • Morino Y., Itoh H. & Wada H. 1963. Crystallization of 2-oxoglutarate L-asparatate transaminases. Biochem. Biophys. Res. Commun. 13: 348–352.

    Article  CAS  Google Scholar 

  • Nagashima F., Tanase S., Fukumoto Y., Joh T., Nomiyama H., Tsuzuki T., Shimada K., Kuramitsu S., Kagamiyama H. & Morino Y. 1989. cDNA cloning and expression of pig cytosolic aspartate aminotransferase in Escherichia coli: aminoterminal heterogeneity of expressed products and lack of its correlation with enzyme function. Biochemistry 28: 1153–1160.

    Article  PubMed  CAS  Google Scholar 

  • Nisselbaum J.S. & Bodansky O. 1966. Kinetics and electrophoretic properties of the isozymes of aspartate aminotransferase from pig heart. J. Biol. Chem. 241: 2661–2664.

    PubMed  CAS  Google Scholar 

  • Obaru K., Tsuzuki T., Setoyama C. & Shimada K. 1988. Structural organization of the mouse aspartate aminotransferase isoenzyme genes. Introns antedate the divergence of cytosolic and mitochondrial isoenzyme genes. J. Mol. Biol. 200: 13–22.

    Article  PubMed  CAS  Google Scholar 

  • Orlacchio A., Scaramuzza E. & Turano C. 1975. Aspartate aminotransferase from wheat germ: purification and kinetic properties. Ital. J. Biochem. 24: 119–137.

    PubMed  CAS  Google Scholar 

  • Ovchinnikov Y.A., Egorov C.A., Abdulaev N.G. & Chashchin V.J. 1975. Primary structure of cytoplasmic aspartate aminotransferase from pig heart muscle. Isolation, purification and characterization of thermolytic peptides. Biokhimiia 40: 634–644.

    PubMed  CAS  Google Scholar 

  • Pan Q.W., Tanase S., Fukumoto Y., Nagashima F., Rhee S., Rogers P.H., Amone A. & Morino Y. 1993. Functional roles of valine 37 and glycine 38 in the mobile loop of porcine cytosolic aspartate aminotransferase. J. Biol. Chem. 268: 24758–24765.

    PubMed  CAS  Google Scholar 

  • Recasens M., Benezra R., Basset P. & Mandel P. 1980. Cysteine sulfinate aminotransferase and aspartate aminotransferase isoenzymes of rat brain. Purification, characterization, and further evidence for identity. Biochemistry 19: 4583–4589.

    Article  PubMed  CAS  Google Scholar 

  • Schmidt J., Scott R.D. & Metzler D.E. 1982. Covalently modi-fied peptides isolated from aspartate aminotransferase after reaction with pyridoxal 5’-phosphate. Biochemistry 21: 5220–5224.

    Article  PubMed  CAS  Google Scholar 

  • Sung M.H., Tanizawa K., Tanaka H., Kuramitsu S., Kagamiyama H. & Soda K. 1990. Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species. J. Bacteriol. 172: 1345–1351.

    PubMed  CAS  Google Scholar 

  • Thompson J.D., Higgins D.G. & Gibson T.J. 1994. CLUSTAL W, improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680.

    Article  PubMed  CAS  Google Scholar 

  • Thornburg J.M., Nelson K.K., Clem B.F., Lane A.N., Arumugam S., Simmons A., Eaton J.W., Telang S. & Chesney J. 2008. Targeting aspartate aminotransferase in breast cancer. Breast Cancer Res. 10: R84.

    Article  PubMed  Google Scholar 

  • Twomey C.M. & Doonan S. 1997. A comparative study of the thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases. Biochim. Biophys. Acta 1342: 37–44.

    Article  PubMed  CAS  Google Scholar 

  • Vessal M. & Taher M. 1995. Partial purification and kinetic properties of human placental cytosolic aspartate transaminase. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 110: 431–437.

    Article  PubMed  CAS  Google Scholar 

  • Wu T.H., Oses-Prieto J.A., Iriarte A. & Martinez-Carrion M. 2003. Release of pyridoxal 5′-phosphate upon unfolding of mitochondrial aspartate aminotransferase. Biochim. Biophys. Acta1647: 315–320.

    CAS  Google Scholar 

  • Zechini B., Pasquazzi C. & Aceti A. 2004. Correlation of serum aminotranferases with HCV-RNA levels and histological findings in patients with chronic hepatitis-C: the role of aspartate aminotransferase in the evaluation of disease progression. Eur. J. Gastroenterol. Hepatol. 16: 891–896.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan

    Muhammad Shahid Nadeem, Naeem Rashid, Muzaffar Iqbal, Qurra-tul-Ann Afza Gardner & Muhammad Akhtar

  2. Calzyme Laboratories, Inc., 3443 Miguelito Court, San Luis Obispo, CA, 93401, USA

    Muzaffar Iqbal

  3. School of Biological Sciences, University of Southampton, Southampton, SO16 7PX, UK

    Muhammad Akhtar

Authors
  1. Muhammad Shahid Nadeem
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Naeem Rashid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Muzaffar Iqbal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qurra-tul-Ann Afza Gardner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Muhammad Akhtar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Naeem Rashid.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shahid Nadeem, M., Rashid, N., Iqbal, M. et al. First cloning and characterization of aspartate aminotransferase from river buffalo (Bubalus bubalis). Biologia 66, 1202 (2011). https://doi.org/10.2478/s11756-011-0125-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.2478/s11756-011-0125-z

Key words

Search

Navigation