Mats Hanson

Ca2+‐ or Mg2+‐Dependent Enzymic ATP Hydrolysis Associated with the Microsomal Fraction of Frog Sciatic Nerves

The microsomal fraction of frog sciatic nerves was found to contain Ca2+‐ or Mg2+‐dependent hydrolytic activity toward different nucleoside di‐and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p‐nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18–24°C in the presence of 1 mm‐Ca2+ or 1 mm‐Mg2+. Ca2+‐ and Mg2+‐ATP hydrolysis displayed pH maxima around 8.0–8.5 and 7.4–8.0, respectively. Vmax values for Ca2+‐ and Mg2+‐ATP hydrolysis were similar: approx. 12 μmol Pi per h per mg protein with a Km value of approx. 0.05 mm. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10‐6–10‐5m) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.

HPLC Separation of Axonally Transported Proteins in Sciatic Nerves

Axonally transported proteins from the sciatic nerve have been separated by an HPLC system consisting of a Model Varian 5040 pump, Varian 401 Integrator and three columns (TSK4000-3000-2000SW) in series. Different eluents have been tested and a highly reproducible separation was obtained in less than 45 minutes. The transported proteins were radioactively tagged and the fractions collected and analysed by scintillation counting. There was a good correlation between molecular weight and eluent time. An on line radioactivity detector system is under developement. The system has a sensitivity for 14C and 35S comparable to scintillation counting.

EFFECTS OF IONOPHORES X-537A AND A23187 ON RAPID AXONAL TRANSPORT IN VITRO

Publisher Summary This chapter describes the effects of ionophores X-537A and A23187 on rapid axonal transport (AXT) in vitro . Ca 2+ could play a role in axonal transport by controlling microtubule (MT) assembly–disassembly and/or activating an ATPase coupled to AXT. Exposure to Ca 2+ greater than normal, or Ca 2+ deprivation, are conditions that both inhibit AXT. However, the former can only be demonstrated at very high external Ca 2+ concentrations, even if the nerve is desheathed. In an attempt to manipulate cellular Ca 2+ levels at physiological Ca 2+ concentrations, the effects of the divalent ionophores X-537A and A23187 on AXT, ultrastructure, high energy phosphates, and Ca 2+ content in frog sciatic nerves is studied. X-537A was found to be a very potent inhibitor of rapid AXT of labeled proteins in vitro . At a concentration as low as 0.09 μM, the amount of transported material was inhibited by about 50% at an unaffected or slightly retarded rate. The number of MT was reduced and the total Ca 2+ content significantly increased at unchanged ATP and CrP levels. Ca 2+ -high Ringer, which does not affect AXT, potentiated the inhibitory effect of X-537A.