Enzymes in Orthopteran Ontogenesis. VI. Autocatalytic Nature of in vivo Formation of Protyrosinase.

Abstract

Northrop considered the autocatalytic formation of pepsin and trypsin from their zymogens as being an instance of protein synthesis. Therefore, since Wrinch's theory described strata of two-dimensional, cyclol-structured lamina, Langmuir and Schaefer point out that protein growth, as with crystal formation, is a determined one and should reasonably be autocatalytic. In the light of these observations, it should be of some general interest to describe the in vivo formation of an enzyme precursor. The present paper is concerned with the reporting of the autocatalytic nature of the formation of protyrosinase within eggs of the grasshopper, Melanoplus differentialis. The preparation and activation of grasshopper egg protyrosinase has been dealt with in a series of papers (Bodine and Boell, Bodine, Allen, and Boell, Bodine and Allen, ) so that here a brief statement of the essential procedures should suffice. Centrifugation of an egg brei prepared in a physiological normal, phosphate buffered (pH 6.8) sodium chloride solution results in the formation of 3 layers, termed A, B, and C respectively, from the centripetal to the centrifugal poles. The lipoidal layer A contains a naturally occurring activator of the protyrosinase which occurs entirely in the cell-free, comparatively voluminous B layer. Layer C consists of the inert egg membranes and structural elements. The brei was always diluted to such a volume that this precursor or inactive form of tyrosinase present in 1.0 cc of fraction B was that derived from 20 eggs irrespective of their particular age. A solution of tyramine hydrochloride was used for substrate in such an amount that during the tyrosinase-catalyzed oxidation to the end product, melanin, 388 mm of oxygen was consumed. The rate of this oxygen uptake was determined in the standard Warburg manometer at 25°C and was expressed as the reciprocal of the time necessary for half completion of the reaction.