In vitro phosphorylation of Paramecium axonemes and permeabilized cells.
This study seeks to identify phosphoproteins in axonemes from Paramecium tetraurelia whose phosphorylation responses to adenosine 3', 5'-cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified axonemes, over 15 bands ranging from Mr greater than 300 kDa to 19 kDa on SDS-PAGE incorporate 32P from adenosine 5'-gamma-[32P]triphosphate (gamma-32P-ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 microM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X-100-permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: 1) a 29 kDa polypeptide whose cAMP-dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and 2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal-sensitive regulators of motility parameters in the Paramecium axoneme.
Hamasaki
T
T
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York.
Murtaugh
T J
TJ
Satir
B H
BH
Satir
P
P
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D002923:Cilia / Q000502:physiology / Q000648:ultrastructure; D000242:Cyclic AMP / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D008868:Microtubule Proteins / Q000032:analysis / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D015388:Organelles / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D010247:Paramecium / Q000378:metabolism*; D010766:Phosphorylation; D015290:Second Messenger Systems
D003902:Detergents; D008868:Microtubule Proteins; D000242:Cyclic AMP; D002118:Calcium