Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-α-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

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The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions.

The Peptide Chains of Human Plasmin MECHANISM OF ACTIVATION OF HUMAN PLASMINOGEN TO PLASMIN

Pyruvate carboxylase. i. nature of the reaction.

Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex

Publisher Summary This chapter discusses the process of isolation, chromatography, structure, and molecular and catalytic properties of bovine pancreatic ribonuclease. At present there are three simple and widely used chromatographic procedures: (1) Hirs base their method on the carboxyl ion exchange resin IRC-50 with 0.2 M phosphate buffer pH 6.45 as the eluting medium. The principal active component of the enzyme preparation is well retarded and is universally referred to as ribonuclease-A. Several poorly resolved faster running peaks are usually seen, the area having the highest activity and running closest to A normally being called ribonuclease B. The ratio of A to B varies with the preparation but may be as high as 10 to 1. (2) Taborsky has described a system based on carboxymethyl cellulose as the exchanger operated in Tris buffer at pH 8 with a sodium chloride gradient. The excellent and adjustable resolution of this system is frequently useful. The principal peak, labeled D by Taborsky, is indistinguishable from ribonuclease-A in the IRC-50 system. (3) Crestfield found chromatography on sulfoethyl Sephadex valuable. Ribonuclease-A may develop heterogeneity during lyophilization and storage. Aggregation appears to occur. A careful study of the preparation problem has been made by Crestfield by using chromatography on Sephadex G-75, and sulfoethyl Sephadex C-25 as well as IRC-50. These authors recommended that RNase-A be stored as a solution in phosphate buffer at –20°C, that salts be exchanged by dialysis or pre-equilibrated Sephadex columns, and that concentration, if necessary, be effected by ultrafiltration. If lyophilization is necessary, it should be carried out from dilute salt-free solution to minimize aggregate formation. The aggregates can be converted to monomers by heating to 60° for a few minutes at neutral pH. The properties of the ribonuclease dimer are also discussed in the chapter.

24 Bovine Pancreatic Ribonuclease

Some factors which affect the enzymatic digestion of ribonucleic acid.

Before proceeding with our experiments relating structure to function in the enzyme bovine pancreatic ribonuclease (RNase) and before attempting to isolate an active fragment of KNase containing an active center (if it exists), we felt it necessary to improve the methods of assaying for RNase. The first part of this presentation, therefore, will deal with the assay procedures employed and with the effects of salts and urea on these procedures and on the substrate, yeast sodium ribonucleate (YRNA). The second part will attempt to provide answers to the following questions relating the structure of the ribonuclease molecule to its enzymatic activity: (1) Are the 4 disulfide bonds necessary for enzyme activity? (2) Is the tightly coiled form of the molecule essential for its activity? ( 3 ) Is the whole, intact protein essential for its catalytic activity?

George Kalnitsky

Papers

Some factors which affect the enzymatic digestion of ribonucleic acid.

The relation of structure to enzymatic activity in ribonuclease

A mechanism for fluoroacetate inhibition

Intracellular distribution of fumarase, aconitase, and isocitric dehydrogenase in rabbit cerebral cortex

The catalysis of the oxidation of some dihydroxybenzene derivatives by various metallic ions