Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation.
Papaya glutamine cyclotransferase (PQC), present in the laticiferous cells of the tropical species Carica papaya, was purified near to homogeneity. Starting from the soluble fraction of the collected plant latex, a combination of ion-exchange chromatography on SP-Sepharose Fast Flow, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purification factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions located on the hydrophobic surface of the enzyme were converted into their S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33000 Da, contains a unique and highly basic polypeptide chain devoid of disulfide bridges as well as of covalently attached phosphate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emission of the plant enzyme. With a lambdamax of emission at 338 nm and a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by surrounding protein atoms. Fluorescence can thus be used to monitor unfolding of this enzyme. Preliminary experiments show that papaya QC is exceptionally resistant to chemical (guanidinium hydrochloride), acid and thermal denaturation. At first sight also, this enzyme exhibits high resistance to proteolysis by the papaya cysteine proteinases, yet present in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity and interest to further investigate how the structure of this plant enzyme is specified.
Zerhouni
S
S
Protein Chemistry Department (CP 609), Faculty of Medicine, Free University of Brussels, Campus Erasme, Route de Lennik, 808, B-1070 Brussels, Belgium.
Amrani
A
A
Nijs
M
M
Smolders
N
N
Azarkan
M
M
Vincentelli
J
J
Looze
Y
Y
D020106:Acrylamide / Q000494:pharmacology; D019881:Aminoacyltransferases / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007840:Latex / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000502:physiology
D006023:Glycoproteins; D007840:Latex; D010940:Plant Proteins; D020106:Acrylamide; D014364:Tryptophan; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase; D003546:Cysteine Endopeptidases; D019791:Guanidine