Pichlo, Magdalena Anna

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Kölner UniversitätsPublikationsServer

AbstractNew life begins with the fusion of an egg with a sperm cell. However, sperm do not encounteran egg by chance. The succesful encounter of gametes is a special challenge in marine animalkingdom. Most marine animals release their gametes into sea. To increase the success rate offertilisation, eggs release chemoattractants to guide the sperm. The directed motion of spermtowards the source of chemoattractants is called chemotaxis. The chemoattractant of the seaurchin  Arbacia  punctulata is  a  short  peptide  called  resact.  The  receptor  for  resact  is  amembrane-bound guanylyl cylase (ApGC). After stimulation with resact, ApGC synthesisesthe second messenger cyclic guanosine monophosphate. I studied the regulation of ApGCactivity by dephosphorylation. ApGC is highly phosphorylated at rest and undergoes rapiddephosphorylation  after  resact  binding.  The  activity  of  ApGC  declines  duringdephosphorylation;  the  enzyme inactivates.  To study the ApGC in more detail,  I  tried toexpress  ApGC  heterologously.  Unfortunately,  the  recombinant  protein  was  notphosphorylated. Studying ApGC regulation in intact sperm, I was able to show that ApGCphosphorylation is essential for its activity. The dephosphorylated ApGC displays no catalyticactivity. The time constant for the dephosphorylation reaction is ca. 280 ms. This matcheswell  with  the  time  constant  of  inactivation.  Remarkably,  far  more  ApGC molecules  aredephosphorylated  at  low  resact  concentrations  than  are  occupied  by  ligand.  Thus  non-occupied  receptors  are  dephosphorylated,  too,  which  could  account  for  an  adaptationmechanism. What is the nature of the phosphatase? The phosphatase activity in sperm doesnot  require  physiological  responses  caused  by  resact  stimulation.  Classical  phosphataseinhibitors  do  not  block  dephosphorylation.  Taken  together  with  the  rapid  time  course  ofdephosphorylation and the  high ApGC density,  there is  good evidence that  the GC itselfdisplays  phosphatase  activity.  Phosphatase  activity  could  be  a  common  feature  of  othermembrane-bound GCs and could help to understand the regulation of this protein family.

Biochemische Untersuchung der Guanylyl Zyklase aus Spermien des Seeigels Arbacia punctulata

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