Search

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and kcat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.

Dopaminochrome formation is catalyzed by commercially available purified peroxidases (EC 1.11.1.7) such as horseradish, lacto- and myelo-peroxidase using dopamine, hydrogen peroxide or promethazine sulfoxide as substrates. A rat brain fraction (RBF) catalyzes a similar reaction and its catalytic power increases after preincubation with hydrogen peroxide/ascorbic acid. The activity of both the purified enzymes and the RBF preparation is inhibited by carnosine and characterized by excess substrate inhibition. The enzymes recognize different substrates but show the highest affinity for dopamine. The RBF fraction is strongly buffered against oxidation by compounds such as glutathione and by bioreductive enzymes such as DT-diaphorase (EC 1.6.99.2) which can use as a substrate menadione or dopaminochrome. The rat brain dopamine peroxidizing activity appeared to be mostly bound to the synaptosomal fraction. The reaction catalyzed by the purified peroxidases was followed by electron spin resonance spectroscopy and, unlike that catalyzed by RBF, was shown to produce the signal of a transient dopamine-o-semiquinone radical.

Lactoperoxidase, when incubated with increasing amounts of promethazine (P) and promethazine sulfoxide (PO) catalyzes the formation of promethazine sulfoxide accompanied by oxygen consumption. An intermediate radical of PO can be detected by electron spin resonance (ESR). Catalase or superoxide dismutase do not inhibit the reaction while dopamine does. The lactoperoxidase-catalyzed formation of dopaminochrome in the presence of hydrogen peroxide is inhibited by P. Both P and PO inhibit acetyl- and butyrylcholinesterase. Purified enzymes were used throughout the study and horseradish peroxidase but not myeloperoxidase had an activity similar to that of lactoperoxidase.

Promethazine sulfoxide was obtained with a quantitative yield in a horse radish peroxidase-catalyzed reaction of promethazine and hydrogen peroxide and was also prepared by direct chemical synthesis. The enzymatic sulfoxidation of promethazine was studied in vitro as a function of pH, promethazine, and hydrogen peroxide concentration. Promethazine sulfoxide inhibits with an apparent K i of 59.7 μM at pH 5.5 the enzymatic reaction, followed spectrophotometrically, polarographically, potentiometrically, and luminometrically. The reaction was also inhibited by ascorbic acid ( K i 26.8 μM) and glutathione ( K i 41,8 μM). The spectrophotometric techniques employed, together with ESR spectrometry, allowed the identification of at least three radical species formed in the course of the reaction. Promethazine sulfoxide is devoid of the antioxidant effect exhibited by promethazine on rat brain synaptosomes. The sulfoxide also lacks photosensitizing action, while retaining the neuroleptic effect of the parent compound.