The effect of octopamine, cyclic adenosine 3',5'-monophosphate and calcium on protein phosphorylation in Schistocerca gregaria central nervous system

Rotondo, Dino (1984) The effect of octopamine, cyclic adenosine 3',5'-monophosphate and calcium on protein phosphorylation in Schistocerca gregaria central nervous system. PhD thesis, University of Glasgow.

Abstract

The effects of octopamine (a putative neurotransmitter) cyclic AMP and calcium on the phosphorylation of protein from the CNS of the desert locust, (Schistocerca gregaria) were investigated. Two main approaches were used to study protein phosphorylation a) Intact cerebral ganglia were incubated with 32Pi and b) Homogenates of cerebral ganglia were incubated with [gamma32P] ATP. The incorporation of P into total protein and specific protein was measured in both cases. The P bound to total protein was measured by precipitating the proteins with TCA and measuring the radioactivity remaining after treatment of the acid insoluble residue with acidified chloroform/methanol and ribonuclease. The P bound to specific proteins was measured by solubilizing the proteins with SDS and separating them by electrophoresis on 10% polyacrylamide/SDS gels followed by autoradiography. In intact cerebral ganglia octopamine (10 -4 M) stimulated an increase in the phosphorylation of total protein within 10 minutes. This subsequently decreased to the control level between 3 and 6 hours. A second octopamine-stimulated increase was observed after 22 hours. The measurement of specific protein phosphorylation after 10 minutes and 22 hours incubation with octopamine revealed that it stimulated the phosphorylation of a protein (Mr 39,000). The phosphorylation of 2 other proteins (Mr 21,000 and 25,000) occured after 22 hours which did not occur after 10 minutes. Octopamine also stimulated the phosphorylation of the Mr 39,000 protein in homogenates of cerebral ganglia but only under conditions which favoured the elevation of cyclic AMP levels prior to the measurement of protein phosphorylation. Dibutyryl cyclic AMP (10e-3M) stimulated the phosphorylation of the Mr 39,000 as well as at least 8 other proteins in intact tissue and the pattern of phosphorylation was similar to that of cyclic AMP in homogenized tissue. Cyclic AMP (10e-5M) stimulated the phosphorylation of many proteins in homogenates including the Mr 39,000 protein within 10 seconds. The major proportion of cyclic AMP-dependent protein phosphorylation resided in the soluble fraction, although phosphorylation of the Mr 39,000 protein occured in both soluble and particulate fractions. Cyclic AMP-dependent protein kinase activity was maximal at a cyclic AMP concentration of 50 muM and half-maximal at 2.5 muM with a Km of 50 muM ATP and its time course reached a maximum within 60 seconds with 10 muM cyclic AMP and 10 muM ATP. Calcium also stimulated total protein phosphorylation in homogenized tissue. A maximum was achieved in the presence of 700 muM EGTA with a CaCl2 concentration of 1 mM and half-maximal at 400 muM. Calcium (1 mM) stimulated the phosphorylation of 3 proteins (Mr 62,000; 54,000 and 31,000) in crude homogenate. In the soluble fraction calcium (1 mM) only stimulated the phosphorylation of the Mr 54,000 protein no stimulation by calcium alone was observed in the particulate fraction. Incubation of the particulate fraction with calcium (1 mM) and calmodulin (10 mug/ml), however, resulted in the phosphorylation of 3 proteins, (Mr 62,000; 54,000 and 31,000). Cyclic AMP-dependent protein kinase inhibitor prevented the cyclic AMP-dependent phosphorylation of all proteins from crude homogenate, soluble and particulate fractions. In addition, it also prevented the calcium-dependent phosphorylation of proteins from the crude homogenate and soluble fraction and calcium/calmodulin-dependent protein phosphorylation from the particulate fraction.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Biochemistry, Neurosciences
• Date of Award: 1984
• Depositing User: Enlighten Team
• Unique ID: glathesis:1984-76521
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 14:14
• Last Modified: 19 Nov 2019 14:14
• URI: https://theses.gla.ac.uk/id/eprint/76521