Biochemical and enzymatic characterization of a partially purified casein kinase-1 like activity from Trypanosoma cruzi.
Two protein kinase activities that use casein as a substrate, Q-I and Q-II, were identified in the epimastigote stage of Trypanosoma cruzi upon chromatography on Q-Sepharose. Q-I was purified further through concanavalin A-sepharose (Q-I*) to remove any trace of the contaminating protease cruzipain. The optimal activity for Q-I* was obtained at pH 8.0, 25 degreesC, 5 mM MgCl(2) and 75 mM NaCl. The size and pI of Q-I* were determined to be 33-36 kDa and 9.6, respectively. When two selective peptide substrates for casein kinases (CKs) (P1: RRKDLHDDEEDEAMSITA for CK1 and P2: RRRADDSDDDDD for CK2) were used, Q-I* was shown to specifically phosphorylate P1. Kinetic studies showed that Q-I* has a K(m) of 5.3 +/- 0.34 mg/ml for casein, 157.6 +/- 5.3 microM for P1 and 35.9 +/- 3.9 microM for ATP. The enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7) or 1-(5-chloroisoquinoline-8-sulfonyl) (CKI-8), two inactivators of mammalian CKs. CKI-7 behaved as a competitive inhibitor with respect to ATP, with a K(I) of 75-100 microM. Treatment with high concentrations of polylysine or heparin also resulted in a significant inhibition of Q-I*. Two well-known activators of mammalian CKs, spermine and spermidine, were also tested. Spermine and spermidine activated Q-I* in a dose-dependent manner. Based on the following characteristics: (1) the ionic strength required for elution from anion-exchange resins; (2) its molecular size and monomeric structure; (3) pI; (4) high level of specificity for P1; (5) inactivation by CKI-7 and CKI-8; and (6) insensitivity to GTP and low concentrations of heparin, we conclude that Q-I* belongs to the CK1 family of protein kinases.
Calabokis
Maritza
M
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela
Kurz
Liliana
L
Wilkesman
Jeff
J
Galán-Caridad
José Manuel
JM
Möller
Carolina
C
Gonzatti
Mary Isabel
MI
Bubis
José
J
D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000254:growth & development
D002364:Caseins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D047388:Casein Kinases