Alkaline ribonuclease from rye germ cytosol

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Science

Background:

• Ribonucleases (RNases) play crucial roles in RNA metabolism and degradation.
• Understanding the specific properties of plant-derived RNases is important for various biotechnological applications.

Purpose of the Study:

• To isolate and characterize an alkaline ribonuclease from rye (Secale cereale L) germ cytosol.
• To elucidate the enzymatic mechanism and substrate specificity of the purified enzyme.
• To investigate potential amino acid residues involved in the enzyme's active site.

Main Methods:

• Isolation and partial purification of alkaline ribonuclease from rye germ cytosol.
• Enzymatic assays using various RNA substrates and synthetic polyribonucleotides.
• Analysis of hydrolysis products to determine cleavage patterns.
• Chemical modification studies to probe active site residues.

Main Results:

• An alkaline ribonuclease (pH optimum 7.6) was purified, showing exclusive endonuclease activity without other nucleolytic functions.
• The enzyme hydrolyzes phosphodiester bonds in RNA, producing nucleoside 2',3'-cyclic phosphates and 3'-phosphates, with a preference for pyrimidine nucleoside 3'-phosphates.
• The enzyme does not depolymerize double-stranded RNA complexes or hydrolyze synthetic purine cyclic nucleotides.
• Photooxidation susceptibility and inhibition by specific reagents suggest tryptophan involvement in the active center.

Conclusions:

• Rye germ cytosol contains a specific alkaline endonuclease with distinct substrate preferences.
• The enzyme's mechanism involves the hydrolysis of RNA phosphodiester bonds, yielding cyclic and 3'-phosphate nucleotides.
• Evidence points to the active participation of a tryptophan residue in the enzyme's catalytic activity.