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Polyamine Regulation of Protein Phosphorylation in the Brain of the Tobacco Hornworm, Manduca sexta
Author(s) -
Birnbaum Mark J.,
Combest Wendell L.,
Bloom Timothy J.,
Gilbert Lawrence I.
Publication year - 1987
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1987.tb05607.x
Subject(s) - manduca sexta , spermine , spermidine , biochemistry , putrescine , polyamine , phosphorylation , biology , protein kinase a , cytosol , manduca , gtp' , protein phosphorylation , microbiology and biotechnology , chemistry , enzyme , insect , ecology
An analysis of the effects of polyamines on protein phosphorylation in cytosolic fractions of the pupal brain of Manduca sexta showed that spermine elicited an increase in casein phosphorylation in a dose‐dependent manner (maximum three‐ to fourfold at 2.0 m M ), whereas spermidine was less effective and putrescine was without effect. In contrast, with phosvitin as the exogenous substrate, higher doses of polyamines, especially spermine, inhibited phosphorylation. High salt conditions abolished the polyamine response. Cytosol protein kinase activity eluted from DEAE‐cellulose at 0.2–0.3 M NaCl. This activity was enhanced in the presence of spermine, and inhibited in the presence of heparin (IC 50 ∼ 30 ng/ml). The enzyme was characterized by a sedimentation coefficient of 6.5S, and a Stokes radius of 49 Å, consistent with a M r of 130,000. Both GTP ( K m , 55 μ M ) and ATP ( K m , 34 μ M ) were utilized as phosphoryl donors ( V max for ATP being four‐fold higher than that observed for GTP). These results indicate the presence in the insect brain of an enzyme very similar to vertebrate casein kinase II. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and autoradiography demonstrated that low concentrations of spermine (100 μ M ) strongly enhanced the phosphorylation of three high‐molecular‐weight cytosolic proteins (305,000, 340,000, and 360,000) localized in the insect nervous system.