Studies on the synthesis of rat brain synaptic membrane proteins

Abstract

The study of the molecular organisation of specialised synaptic structures has been considerably advanced by the development of methods for the isolation of synaptic subfractions enriched in synaptic plasma membranes (SPM), synaptic junctional complexes (SJC's) and postsynaptic densities (PSD's). The present thesis deals with one important aspect of this organisation, namely the synthesis of SPM proteins and its modulation by selected chemical agents. SPM, mitochondrial and myelin-enriched fractions of comparable purity to published data have been isolated from adult rat brain. An in vivo35method for monitoring incorporation of L-( S)methionine into the protein of these fractions has been developed. This, involved injection of 35 flooding concentrations of L-( S)methionine into the lateral ventricle via a pre-implanted cannula. Since the precursor specific activity is known and constant for at least 45 minutes the incorporation rates can be reliably measured over this period. The effects of two anaesthetics, halothane and pentobarbitone, on brain protein synthesis were investigated. Halothane (3%) produced a gross inhibition of protein synthesis in total forebrain and SPM, myelin and mitochondrial fractions. Pentobarbitone produced no such inhibition at doses of 60 or 75 mg/Kg body weight. L-DOPA was also found to inhibit incorporation rates by close to 30% in total forebrain and mitochondrial protein in hyperthermic animals (body temperature 39-40°C). Other subcellular fractions were less affected by the drug. Thyroid hormone deficiency during brain development impairs brain structure and function including synapse formation. The present study showed that hypothyroidism decreased protein yields in SPM, myelin and mitochondrial fractions and impaired synthesis of SPM and myelin protein. The rates of incorporation into total forebrain and mitochondrial protein were not significantly affected. SDS polyacrylamide gel, electrophoresis and fluorographic studies tentatively showed that the relative rates of synthesis of specific polypeptides were altered in thyroid deficient animals.