Properties of neurofilament protein kinase.
Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 X 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at -20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.
Toru-Delbauffe
D
D
Pierre
M
M
Osty
J
J
Chantoux
F
F
Francon
J
J
D000818:Animals; D047388:Casein Kinases; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D003599:Cytoskeleton / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007382:Intermediate Filaments / Q000187:drug effects / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D009419:Nerve Tissue Proteins; D011494:Protein Kinases; D047388:Casein Kinases; D008274:Magnesium