This study was undertaken to characterize the enzymatic properties of the particulate guanylyl cyclase previously shown to be present at a high level of activity in purified rat brain myelin. Significant activation was achieved by both Lubrol-PX and Triton X-100, the latter being somewhat more effective. A pH optimum of 7.8 was observed, compared to 7.4 for microsomes. Employing 1.2 mM GTP with 1% Triton X-100, linearity of response was observed up to 60 min and approximately 1.2 mg of myelin protein. Kinetic analysis revealed Km values of 0.258mM and 0.486mM for myelin and microsomes, respectively, similar values being obtained by Lineweaver-Burke analysis or Direct Linear Plot. Vmax values were 20 and 266 pmol/mg protein/min for myelin and microsomes, respectively. Washing of the myelin with 0.5 M NaCl or 0.1% Na taurocholate did not remove a significant amount of guanylyl cyclase activity, indicating the enzyme to be intrinsic to the myelin sheath.

The tRNA mediated, posttranslational, N-terminal arginylation of proteins occurs in all eukaryotic cells. In nervous tissue, these reactions can be inhibited by endogenous molecules with a molecular weight of between one thousand and five thousand. In the present experiments, exogenous serine protease inhibitors (10(-5) M or less) but not other types of protease inhibitors, were found to be able to block the arginylation of protein in extracts of rat brain homogenates. Inhibition was not by the usual mode of action of protease inhibitors, but by interfering (non-competitively) with the charging of tRNA. Since arginylated proteins are rapidly ubiquitinated and degraded by cytosolic proteases, serine protease inhibitors may act to stabilize proteins by a dual mechanism of inhibiting arginylation as well as inhibiting serine proteases.

All eukaryotic cells contain enzymes that are able to catalyze the transfer of Arg from tRNA to the N-terminus of naturally short lived or damaged cytosolic proteins. For certain test proteins, it has been shown that the addition of Arg to the N-terminus leads to their degradation via the ubiquitin proteolytic pathway. The mechanisms used by cells for identifying proteins for arginylation and regulating arginylation are not known. The present study reports the isolation of a peptide from rat brain that is able to inhibit the arginylation of proteins in brain extracts. We suggest that this peptide is the physiological regulator of arginylation in rat brain.

No abstract

No abstract