Cyclic nucleotide-independent phosphorylation of vitellin by casein kinase II purified from Rhodnius prolixus oocytes.
In this study we show that Vitellin (VT) phosphorylation in chorionated oocytes of Rhodnius prolixus is completely inhibited by heparin (10 microg/ml), a classical casein kinase II (CK II) inhibitor. VT phosphorylation is not affected by modulators of cyclic nucleotide-dependent protein kinases such as c-AMP (10 microM), H-8 (1 microM) and H-89 (0.1 microM). We have obtained a 3000-fold VT-free enriched preparation of CK II. Autophosphorylation of this enzyme preparation in the presence of (32)P-ATP demonstrated that it lacks any endogenous substrates. Rhodnius CK II is strongly inhibited by heparin (Ki = 9 nM) and uses ATP (Km = 36 microM) or GTP (Km = 86 microM) as phosphate donors. Incubation of VT with purified Rhodnius CK II and (32)P-ATP led to the incorporation of 2 mols of phosphate/mol VT. However, the total number of phosphorylation sites available can be altered by previous incubation of VT with alkaline phosphatase. These data show that an insect yolk protein contain phosphorylation sites for a cyclic nucleotide-independent protein kinase such as CK II.
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, UFRJ, Cidade Universitária, Rio de Janeiro, RJ, CEP 21941-590, Brazil. maneto@bioqmed.ufrj.br
Mário A C
MA
Silva-Neto
Eliane
E
Fialho
Márcia C
MC
Paes
Pedro L
PL
Oliveira
Hatisaburo
H
Masuda
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D047390:Casein Kinase II; D000242:Cyclic AMP / Q000378:metabolism; D004527:Egg Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D066298:In Vitro Techniques; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012239:Rhodnius / Q000378:metabolism*
D004527:Egg Proteins; D004791:Enzyme Inhibitors; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006493:Heparin; D000242:Cyclic AMP; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases