Membrane bound γ-glutamyltranspeptidase: Its structure, biosynthesis and degradation
ABSTRACT γ-Glutamyltranspeptidase is a glycoprotein composed of heavy and light subunits and associated with the brush border membrane of the kidney and small intestine. γGTP solubilized with papain is a hydrophilic enzyme which has lost the membrane binding segments but its catalytic activity is not altered, whereas γGTP solubilized with Triton X-100 is a hydrophobic enzyme which contains hydrophobic domain binding to the membrane. Amino acid compositions of these two forms were compared and Triton solubilized enzyme was found to contain 52 amino acid residues more than the papain solubilized form. This difference is due to the heavy subunit not light subunit. Then, end group analysis was carried out and the carboxyl-termini of their light subunits were found to be phenylalanine and those of their heavy subunits were tyrosine, respectively. Although light subunits of two forms contain a common sequence, Thr-Ala (X)-Leu as an amino-terminal portion, that of heavy subunit of Triton X-100 solubilized form contains the sequence Met-Lys-Asn-Arg-Phe-Leu-Val-Leu-Gly-Leu-Val-Ala-Val-Val-Leu-Val-Phe-Val-Ile-Ile-Gly-Leu and the papain-solubilized form contains completely different amino-terminal sequence Gly-Pro-Pro-Leu. It is concluded that an amino-terminal portion of the heavy subunit is a hydrophobic domain consisting of about 20 hydrophobic amino acids and contributes to anchor the enzyme to the membrane. γGTP has been known to show great heterogeneity in charge and multiple forms with different isoelectric points are found to be mainly due to differences of their sugar chains. Then the structures of the oligosaccharides attached to γGTP were determined. They were found to be all asparagine linked and consisted of neutral and acidic oligosaccharides with remarkable heterogeneity. A correlation between the contents of the acidic oligosaccharides and the isoelectric points of multiple forms of γGTP was observed. In addition, multiple forms of γGTP were immunologically identical and their protein structures were identical.Next, the mechanisms of biosynthesis of γGTP were examined and it was found that two subunits of γGTP are synthesized as a precursor protein with a single polypeptide chain of 78,000 daltons. Then processing by limited proteolysis occurs post-translationally, and it is a rather slow process. Since the precursor form is already core glycosylated and fucosylated, proteolytic processing could be carried out after completion of terminal glycosylation at the Golgi membrane or the plasma membrane. Moreover, the turnover rates of the heavy and light subunits were the same with half lives of 4.3 days. Thus, although two subunits of γGTP bear a different function, they are synthesized and degraded together. On the other hand, γGTP and aminopeptidase M are closely associated with the microvillus membrane of kidney and small intestine, whereas the two enzymes turn over independently. It suggests that biosynthesis and the degradation of the microvillus enzymes are not carried out as a membrane unit; each enzyme has its own turnover rates.
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ABSTRACT: Two-dimensional electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or specific immunoblotting analysis, was applied to the characterization of gamma-glutamyltransferase (GGT, EC 184.108.40.206.) in rat tissues. We confirmed that purified kidney GGT is a dimer composed of two non-identical subunits with molecular masses of about 50 and 30 kDa. Both the light and the heavy subunits were separated into 6 and 2 protein bands, respectively. Antibody fractions against the 30 and 50 kDa subunits, purified by immunoaffinity from a whole antiserum directed to the rat kidney enzyme, recognized their corresponding subunits and did not cross-react with each other. Studies of the reactivity of these antibodies towards GGTs from kidney and liver homogenates revealed an intraspecies dissimilarity in the molecular architecture of the kidney and liver GGTs, especially concerning the 30 kDa subunit. Following phenobarbital treatment of animals we observed an increase in immunoreactive GGT, accompanied by the appearance of additional polypeptides with more basic isoelectric points and higher molecular mass.Electrophoresis 01/1986; 7(2):83-88. · 3.26 Impact Factor
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ABSTRACT: The role of gamma-glutamyltransferase (gamma-GT) in renal ammoniagenesis and glutamine utilization was evaluated in the intact functioning rat kidney. Total NH4+ released, as the sum of renal venous and urinary NH4+, was measured under conditions of chronic metabolic acidosis and paraminohippurate infusion. Ammonia derived from extracellular gamma-GT hydrolysis of glutamine was differentiated from that produced by intracellular phosphate dependent glutaminase (PDG) by employing acivicin, a gamma-GT inhibitor. In non-acidotic animals acivicin administration inhibited gamma-GT 95% and renal venous NH4+ release 48%; NH4+ release into the urine was not inhibited. Chronic metabolic acidosis elevated total NH4+ release 2.5fold, associated with adaptive increase in both gamma-GT and PDG; acivicin reduced total NH4+ released 36% with both renal venous and urinary release effected. The contribution of gamma-GT to total NH4+ production doubles in metabolic acidosis in agreement with the adaptive rise in the in vitro assayed gamma-GT activity. Luminal ammoniagenesis increases in chronic acidosis associated with a fall in urinary glutamine concentration and a rise in the blood to urine glutamine concentration gradient; gamma-GT inhibition eliminates this gradient suggesting luminal ammoniagenesis is largely dependent upon the paracellular glutamine flux. In support of this, paraminohippurate (PAH) infusion increased total renal NH4+ release due entirely to enhanced NH4+ excretion. PAH stimulated luminal ammoniagenesis was associated with an acceleration of renal glutamine extraction and a steeper blood to urine glutamine diffusion gradient; acivicin blocked this response consistent with PAH secretion coupled to activation of intraluminal gamma-GT and glutamine hydrolysis.Pflügers Archiv - European Journal of Physiology 06/1988; 411(5):573-8. · 4.87 Impact Factor
- BioFactors 02/2003; 17(1-4):161-73. · 3.09 Impact Factor