The Isolation and Properties of Cardiac Ribosomes and Polysomes

Abstract

1. A method is described by which good yields of ribosomes and polysomes free of contamination by submitochondrial fragments can be prepared from rat cardiac muscle. These preparations are capable of incorporation of amino acids into protein in vitro. 2. The ribosome preparation consists of 32% of monomeric ribosomes and 68% of ribosomal aggregates or polysomes. The polysome preparation has a decreased monomeric content. Dimers, trimers, tetramers, pentamers and larger components can be differentiated. 3. The polysome aggregate structure is degraded to monomeric ribosomes on incubation with small amounts of ribonuclease or by preparation in the absence of Mg(2+) ions. The degradation in the absence of Mg(2+) ions was not reversible and drastically decreased the incorporation of amino acids in vitro. 4. The cardiac ribosomes contained two major RNA species sedimenting at 19s and 28s in a 1:2.4 ratio. 5. The RNA/protein ratio of cardiac ribosomes and polysomes was consistently lower than that of similar preparations from liver. The concentrations of Na(+) and K(+) ions present during preparation had a great effect on the RNA/protein ratio. 6. Optimum conditions for the incorporation of amino acids into protein in vitro are reported. Cardiac ribosomes have a lower rate of incorporation of amino acids in vitro than liver ribosomes. 7. Heart cell sap is less active than liver cell sap: evidence is presented that a factor, present in liver cell sap and concerned with stimulating the synthesis of the peptide chain, is lacking in heart cell sap. 8. Pulse-labelling of perfused hearts followed by examination of the subcellular structures showed that the ribosomal fraction was the most active in the incorporation of amino acids in vitro.

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte

“…NADPH-cytochrome c oxidoreductase activity (EC 1.6.2.4) was assayed by the method of Sottocasa et al (1967). Succinate dehydrogenase activity (EC 1.3.99.1) was assayed by the procedure of Earl & Korner (1965). Monoamine oxidase activity (EC 1.4.3.4) was determined spectrophotometrically as described by Schnaitman et al (1967).…”

An azide‐insensitive low‐affinity ATPase stimulated by Ca2+ or Mg2+ in basal‐lateral and brush border membranes of kidney cortex

“…Succinate dehydrogenase activity was measured at 37 C a s described by Earl and Korner [17]. The succinate dependent reduction of 2,6-dichloroindophenol was monitored at 600 nm.…”

“…2A. The basal-lateral membrane fractions (10)(11)(12)(13)(14)(15)(16)(17) were pooled and re-electrophorized, to eliminate contaminating brush border membranes (Fig. 2 B).…”

Correlation between renal membrane binding and nephrotoxicity of aminoglycosides

“…Na+,K+-ATPase was measured by a linkedenzyme assay in which oxidation of NADH was continuously monitored at 340 nm in the presence or absence of 1 mM ouabain (35). Alkaline phosphatase was measured at 420 nm by the p-nitrophenyl phosphate method (27), and succinic dehydrogenase was monitored at 600 nm by a method described by Earl and Korner (9). HRP was assayed by a colorimetric method with hydrogen peroxide and pyrogallol as substrates (39).…”