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Kinetic studies of adenylyl cyclase of fat cell membranes

II. Comparison of activities measured in the presence of Mn++-AMP-P(NH)P and Mg++-AMP-P(NH)P Effects of insulin, fluoride, isoproterenol, and GMP-P(NH)P

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Summary

The kinetics of fat cell adenylyl cyclase were studied, with AMP-P(NH)P and Mn++ or Mg++ as the divalent cation. In general, the reaction times were not linear. In the presence of fluoride or GMP-P(NH)P, the time curves were concave upwards; in other cases (i.e., basal activity, insulin, or isoproterenol), transient rates tended to decrease with time during the assay. Kinetic data were analyzed according to a previously described procedure (Torreset al., 1978b) which isolates two kinetic components: initial and final.

With AMP-P(NH)P, kinetic activities were about ten times lower than those for ATP. With Mn++, activities were at least two-times higher than for Mg++.

Spontaneous inactivation of adenylyl cyclase was higher in assays containing Mg++ than in those supplemented with Mn++. In the latter case, insulin was able to increase the inactivation rate. Fluoride and isoproterenol both activated adenylyl cyclase in both the initial and final kinetic components; under most of the conditions explored, their effects on the final component appeared to be more dramatic. Assays with GMP-P(NH)P showed inhibited activity in the initial component and increased activity in the final one.

When the results obtained with AMP-P(NH)P are compared with those of ATP (Torreset al., 1978b. J. Membrane Biol. 43:000), the following differences were found: (i) in the presence of insulin and Mn++, cyclase inactivation was higher with AMP-P(NH)P than with ATP; (ii) fluoride stimulation of the final component was more marked with ATP than with AMP-P(NH)P; (iii) cyclase stimulation by isoproterenol was slightly higher with the nucleotide analog; and (iv) GMP-P(NH)P stimulation of the final component resulted in higher activity with ATP than with AMP-P(NH)P.

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Author notes

  1. H. N. Torres & M. M. Flawiá

    Present address: Fundacion Campomar, Instituto de Investigaciones Bioquimicas, Obligado 2490, 1428, Buenos Aires, Argentina

  2. J. A. Medrano

    Present address: Department of Physics, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina

Authors and Affiliations

  1. Department of Molecular Biology, The Wellcome Research Laboratories, 27709, Research Triangle Park, N.C.

    H. N. Torres, M. M. Flawiá, J. A. Medrano & P. Cuatrecasas

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  1. H. N. Torres
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  4. P. Cuatrecasas
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Torres, H.N., Flawiá, M.M., Medrano, J.A. et al. Kinetic studies of adenylyl cyclase of fat cell membranes. J. Membrain Biol. 43, 45–69 (1978). https://doi.org/10.1007/BF01869041

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  • DOI: https://doi.org/10.1007/BF01869041

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