Thomas Hunter Allen

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

Northrop 1 considered the autocatalytic formation of pepsin and trypsin from their zymogens as being an instance of protein synthesis. Therefore, since Wrinchs 2 theory described strata of two-dimensional, cyclol-structured lamina, Langmuir and Schaefer 3 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, 4 Bodine, Allen, and Boell, 5 Bodine and Allen 6 , 7 ) 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 3 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.

Protyrosinase and Polar-Nonpolar Cations and Anions.

The sodium alkyl sulfates, which activate protyrosinase, 1 yield polar-nonpolar anions, (RSO4). Alkylamine hydrochlorides, however, give polar-nonpolar cations (RNH3.H+), and do not activate protyrosinase. † This distinction may be of value in the search for the kind of chemical group or part of protyrosinase which is involved in its activation. Three similar preparations of protyrosinase were extracted from grasshopper eggs (Melanoplus differentialis) by a method involving trituration, fractional precipitation, and dialysis. 1 Activities, measured in a Warburg apparatus, were compared by the reciprocal—multiplied by 1000—of the time in minutes for the first 100 μ1 of oxygen uptake with tyramine hydrochloride as substrate. The ascending limb of curve A (Fig. 1) shows the activation of protyrosinase that occurs in the presence of sodium dodecyl sulfate. With dodecylamine hydrochloride (Curve F, Fig. 1) there seems to be no conversion of protyrosinase into tyrosinase. When solutions of alkyl sulfates and alkylamines are mixed, a precipitate forms. Thus, from activation experiments performed by adding a constant amount of protyrosinase to such mixtures (Curves B, C, D, E, Fig. 1), it was found at half activation that 1.00 mole of either decylor dodecylamine hydrochloride removes 0.36, 1.01, and 2.79 moles of sodium octyl, decyl, and dodecyl sulfates (Fig. 2). These values may well be a function of the dissociation constants of the compounds formed by cations and anions. The effects of variation in the order of mixing of reagents is graphically illustrated in Fig. 3. The upper curve (A) shows only a slight decrease in tyrosinase when excess dodecylamine is added after the mixing of protyrosinase and dodecyl sulfate. Curve B describes the effect, previously noted, of mixing dodecyl sulfate with dodecylamine and, after equilibration, adding the protyrosinase. Curve C refers to the results of mixing protyrosinase and dodecylamine and then adding the dodecyl sulfate.