Ribonuclease succinylation

The smallest member of a new family of prolyl iso-merases (unrelated to the cyclophilins or the FK-506 binding proteins) that catalyzes the proline-limited folding of a variant of ribonuclease T1 with a KJK value of 30,000 M s With the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide as a substrate in parvulin-catalyzed prolyl isomerization, this parameter is 1.1 x 10 M s Parvulin also accelerates its own refolding in an autocatalytic fashion. 

Incubation of succinylated yeast homogenate at 55°C (pH 6.0). to activate endogenous ribonuclease and hydrolyze the contaminant RNA resulted in the significant reduction of the extensive proteolysis normally observed (Table VI). Furthermore, the coagulation and precipitation of protein that normally occurs during this step was also eliminated. 

Thus, succinylation prevented the aggregation and coagulation of yeast proteins normally observed during RNA hydrolysis with endogenous ribonuclease and markedly increased the recovery of intact, soluble protein. Lindbloom (69) reported that up to 70% of yeast protein may be degraded during incubation to reduce RNA (pH 6,... 

Because derivatization impaired proteolysis, its effect on endogenous ribonclease was studied. Ribonuclease activity was inhibited with increasing succinylation and dropped sharply at an anhydride to protein ratio of 0.8 where 80% of e-NH2 groups of the total protein were succinylated (Fig. 5). Because the ribonuclease was inactivated, an increased nucleic acid content in the precipitated proteins was expected. However, we observed less nucleic acid (NA) in precipitated succinylated protein than in the non-modified controls. Maximum precipitation of succinylated protein occurred around pH 4.5 and contained only 1.8% nucleic acid on a... 

Figure 5. The effects of succinylation during extraction of yeast protein on the endogenous ribonuclease activity (pH 6.0, 55°C)...

The absorption spectra of three yeast protein preparations prepared by different procedures were compared (Fig. 8). The presence of nucleic acid which has a X maximum at 260 nm tend to shift the absorption spectrum of yeast protein to lower wavelengths. The ratio of absorption at 280 to 260 nm is indicative of NA contamination in protein samples a ratio of more than one indicates pure protein devoid of nucleic acid whereas a ratio of 0.65 indicates approximately 30% contamination with NA. The yeast protein extracted with alkali and directly acid precipitated showed a X max at 260, a 280/260 ratio of 0.67 and contained 28%, NA determined chemically. Protein extracted in alkali, adjusted to pH 6 and incubated at 55°C for 3-5 hours, to reduce NA with endogenous ribonuclease, had a X max at 260, a 280/260 ratio of 0.8 and a NA content of 3.3% while yeast protein prepared by the succinylation procedure and precipitated at pH 4.5 showed a X max at 275 nm, a 280/260 ratio of 1.0 and nucleic acid content of 1.8. 

Legend crude protein prepared by precipitation of an alkali extract at pH 4.0 ( ) yeast protein obtained following activation of endogenous ribonuclease (82) (O), and yeast protein prepared by the succinylation procedure (O). 

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