ABSTRACT
This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at -20 degrees C. The total protein extract was isolated from 5 samples in each case (mature and hyper mature cataract) and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 degrees C. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. beta mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates.
Subject(s)
Aged , Humans , Middle Aged , Amino Acids , Metabolism , Cataract , Pathology , Endopeptidases , Metabolism , Enzyme Activation , Hydrogen-Ion Concentration , Ions , Chemistry , Metals , Chemistry , Pharmacology , Substrate SpecificityABSTRACT
Mulberry longicorn beetle, Apriona germari, has been reported to produce two endo-beta-1,4-glucanases or AgEGases (accession Nos. Q6SS52 and Q5XQD1). AgEGase sequence contains catalytic motif (amino acid residues 37-48), which is the characteristic of family Glycohydrolase 45 and is identified as the substrate binding site. The application of bioinformatics approaches includes sequence analysis, structural modeling and inhibitor docking to relate the structure and function of AgEGases. We have dissected the sequence and structure of AgEGase catalytic motif and compared it with crystal structure of Humicola insolens endoglucanases V. The results show an involvement of sulfur containing amino acid residues in the active site of the enzyme. Cys residues and position of disulfide bonds are highly conserved between the two structures of endoglucanases of A. germari. Surface calculation of AgEGase structure in the absence of Cys residues reveals greater accessibility of the catalytic site to the substrate involving Asp42, a highly conserved residue. For the inhibition study, tannin-based structure was docked into the catalytic site of AgEGase using ArgusLab 4.0 and it resulted in a stable complex formation. It is suggested that the inhibition could occur through formation of a stable transition state analog-enzyme complex with the tannin-based inhibitor, as observed with other insect cellulases in our laboratory.
