Intraneural Mitochondria: INCORPORATION OF AMINO ACIDS AND MONOSACCHARIDES INTO MACROMOLECULES BY ISOLATED SYNAPTOSOMES AND SYNAPTOSOMAL MITOCHONDRIA

Abstract

Abstract Glycoprotein, glycolipid, protein, and lipid synthesis was measured in isolated sterile synaptosomes and sterile intraneural mitochondria by the incorporation of radioactive precursors into endogenous macromolecules. The purity of two synaptosomal preparations and three intraneural mitochondria preparations from male guinea pig cortices was assayed by marker enzymes. The preparation of intraneural mitochondria used in the experiments was free of nonmitochondrial (contaminating) enzymes and showed a high specific activity of the mitochondrial enzymes monoamine oxidase and succinic dehydrogenase. Leucine and glycine and, to a lesser extent, aspartic acid were incorporated into protein by both synaptosomes and intraneural mitochondria. Monosaccharides from UDP-glucose, UDP-galactose, GDP-mannose, and glucosamine were incorporated into both glycoprotein and glycolipid by the isolated synaptosomes and intraneural mitochondria but no incorporation of UDP-xylose or UDP-arabinose occurred. Chloramphenicol at levels of 83 µg per ml of incubation mixture inhibited protein synthesis in the intraneural mitochondria to 60% of normal activity; higher levels caused even greater inhibition. Cycloheximide at 0.83 mg per ml inhibited protein synthesis in the intraneural mitochondria to about 70% of normal activity; 6.66 mg per ml caused inhibition to 40% normal activity. Glycoprotein synthesis was inhibited in the intraneural mitochondria by both antibiotics but to a lesser extent than protein synthesis. Glycoprotein and glycolipid synthesis in the intraneural mitochondria showed a marked requirement for divalent cations, with Mn++ being the best activator; some ions substituted for Mn++ but others were inhibitory to incorporation. Neither glycoprotein nor protein synthesis was adversely affected by RNase in either synaptosomes or the intraneural mitochondria. Identification of the radioactive products indicated some epimerization of the sugars before incorporation into the macromolecules. The reactions for glycoprotein and glycolipid synthesis showed broad temperature dependence with optimum temperatures of about 37°. The reactions were linear with respect to time for only 15 to 45 min.