Modification of fructose bisphosphatase by a proteolytic enzyme from rat liver lysosomes

Abstract

Membrane preparations derived from the lysosome-rich heavy particle fraction from rat liver contain a proteinase (converting enzyme) that increases the activity of rabbit liver fructose 1,6-bisphosphatase, assayed at pH 9.2, by approximately six-fold. The activity assayed at pH 7.5 is slightly decreased. These changes are associated with the hydrolysis of several peptide bonds in the tetrameric enzyme (subunit molecular weight =36,000). Initially, hydrolysis appears to occur at either of two distinctly different peptide bonds, yielding modified subunits having molecular weights of approximately 29,000 and 26,000, respectively. The larger modified subunit is resistant to further degradation, but the 26,000 subunit is hydrolyzed to fragments whose mass is approximately 13,000 daltons. The cleaved protein retains its original size and tetrameric structure, but is dissociated into its component peptides on treatment with sodium dodecyl sulfate or during carboxymethylation in urea. One of the sites of hydrolysis has been identified by sequence analysis as the peptide bond between residues Asn 64 and Val 65 ; cleavage of this bond yields the 29,000 subunit and a 6,700 dalton peptide containing the NH 2 -terminus. This bond is adjacent to one of the sites of cleavage by subtilisin reported earlier, supporting the view that in the tetrameric protein each subunit possesses an exposed peptide region that is susceptible to proteolytic attack.