Preparation of peroxisomes from carp liver by zonal rotor censity gradient centrifugation

Synopsis Peroxisomes from carp liver can be separated by isopycnic density gradient centrifugation in sucrose. Without reaching complete sedimentation equilibrium, the purification by this method is quite successful. There is a 40-fold enrichment of catalase, the peroxisomal marker, with a total yield of 27%. No pretreatment of animals is necessary for separation from lysosomes, which, besides high fragility, show lower buoyant densities than peroxisomes. The enzyme content of carp liver peroxisomes is similar to that of rat liver, with the exception of -glycerophosphate dehydrogenase, which in this tissue is a completely soluble cytoplasmic enzyme. Total activities are much lower than in the rat, for the characteristic peroxisomal oxidases the difference being in the range of one order of magnitude.     

A study of nucleated erythrocytes by density-gradient centrifugation in a zonal rotor

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [(32)P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35mu(3) and are isopycnic with sucrose of density 1.2871 (measured at 20 degrees ). Maturation of the nuclei causes them to shrink to a volume of 25mu(3) and the density to increase to 1.2944.     

Separation of plant cell organelles by zonal centrifugation in reorienting density gradients

Preparative separations of plant cell organelles (glyoxysomes, proplastids, mitochondria, and endoplasmic reticulum) in an ordinary refrigerated centrifuge were obtained by sucrose density gradient centrifugation in a zonal rotor loaded and unloaded in the static manner. The quality of the separation which was monitored by marker enzymes and electron microscopy compares to analytical separations in swinging-bucket rotors. Membrane alterations observed in glyoxysomes and mitochondria are traced back to sucrose as a major component of the homogenization and density gradient medium.     

The simultaneous measurement of densities and absorbances in sucrose gradients, applied to zonal centrifugation

An apparatus is described that continuously and simultaneously registers both densities and absorbances. An application for centrifugation with a zonal rotor is given.     

The purification of cell nuclei from calf uterus by zonal centrifugation in reorienting density gradients

A method is described for isolation of substantial amounts of pure and enzymatically active nuclei from whole calf uterus. The technique involves a multistep sequential homogenization of the tissue and a zonal centrifugation of the crude nuclear preparation in a reorienting density gradient rotor. Electron and phase contrast microscopic observations show that the nuclei are intact and practically free from cytoplasmic contamination. Based on DNA recovery, the purified fraction contains 9% of the nuclei of the total tissue and more than 19% of the filtered homogenate. The pure nuclear fraction consists of 29% DNA, 7% RNA, and 64% protein, which parallels the composition of purified nuclei from other mammalian tissues.     

Distribution of selected phospholipid modifying enzymes in rat brain microsomal subfractions prepared by density gradient zonal rotor centrifugation

A rat brain P₃ fraction enriched in ER derived microsomes was centrifuged through a 20–40% linear sucrose gradient in a Beckman Ti-14 Zonal rotor and 11 fractions were obtained. The distribution of marker enzyme activities and protein were determined in these 11 subfractions. NADPH-Cytochrome C reductase, choline phosphotransferase were employed for endoplasmic reticulum, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were employed for plasma membrane, 2, 3-cyclic nucleotide phosphohydrolase was employed for myelin. The bulk of the protein was recovered in the 24–34% sucrose fractions, Na⁺, K⁺-ATPase, 5-nucleotidase, and acetylcholinesterase were in the 22–38% sucrose fractions while NADPH-cytochrome C reductase and CNPase were enriched in the 20–22% sucrose fractions. The ethanolamine and the serine base exchange activities had a bimodal distribution, with highest specific activities in sucrose fractions 32–34% and 20–24%. Choline base exchange activity was nearly undetectable in all the fractions. The specific activities of CDP-choline phosphotransferase, and phospholipid-N-methyltransferase were highest in the 20–22% sucrose fraction. Phospholipid-N-methyltransferase activity was significantly stimulated in the presence of exogenous phospholipid acceptors as phosphatidylethanolamine or phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine, however, the greatest response was with phosphatidylmonomethylethanolamine. The rat brain P₃ fraction yielded a population of a membrane at the light end of the sucrose gradient which has a buoyant density similar to myelin but seemed to be enriched with NADPN cytochrome C reductase and phospholipid modifying enzymes. This is in contrast to liver microsomes submitted to a similar fractionation.     

Generation of density gradients. Approximations to equivolumetric gradients for zonal rotors

The use of a “density gradient generating function” allows the concentration profile of a density gradient to be written explicitly in terms of the required distribution of sedimentation coefficients in place of the previous implicit formulations. This function, which is easily implemented in a computer program, permits calculation of density gradients for a number of applications. This approach is applied to computation of a variety of equivolumetric gradients of sucrose for zonal rotors and yields a formula for the calibration of such gradients. An accurate approximation has been found which allows the generation of virtually all equivolumetric gradients of sucrose for a given rotor using a single program for the gradient generator employed; the adjustment for different particle densities and for different concentrations at the top of the gradient is made by varying only the initial and final concentrations of sucrose used.     

Oxidative phosphorylation in rat liver mitochondria isolated by rate zonal centrifugation: Examination of Ficoll gradients and subpopulations of mitochondria

In order to measure the parameters of oxidative phosphorylation it is necessary to isolate physiologically intact mitochondria. The isolation of rat liver mitochondria by rate zonal centrifugation utilizing isoosmotic Ficoll gradients resulted in the uncoupling of oxidative phosphorylation in these organelles. Analysis of the Ficoll solutions used to construct the gradients indicated that the Ca²⁺ content (200–400 nmole Ca²⁺/mg protein) was sufficiently high to cause an uncoupling of oxidative phosphorylation. Treatment of the Ficoll solutions with Amberlite MB-3 resin reduced the Ca²⁺ content to levels below the limit of determination of the assay procedure. This resulted in the retention of respiratory control (1.42) in rate-zonally centrifuged mitochondria. The addition of bovine serum albumin (100 mg%) to the Ficoll gradients increased the respiratory control index to 2.10. The increase is due to an elevation in state 3 respiration rather than any change in state 4 respiration. The addition of 200 mg% bovine serum albumin to the Ficoll gradient did not further enhance the respiratory control index.Examination of subpopulations of rat liver mitochondria revealed that they are heterogeneous with regard to states 3 and 4 respiration, respiratory control indices, and ADP:O ratios. In mitochondrial subpopulations respiratory control indices ranged from 1.00 to 4.13 and ADP:O ratios ranged from 1.22 to 1.83. This investigation defined a procedure for the isolation of physiologically intact mitochondria from rat liver homogenates.     

Macro and micro rate zonal analytical centrifugation of polydisperse and slowly diffusing sedimenting systems in isovolumetric density gradients. Application to cartilage proteoglycans

A method to study the polydispersity of zonally sedimenting and slowly diffusing macromolecules or particles in isokinetic or isovolumetric density gradients is presented. First, a brief theory is given for predicting the zonal profile after a "triangular" (or "inverse") zone is centrifuged. This type of zone is essential to preserve hydrodynamic stability of the very slowly diffusing polydisperse solutes. It is proven, both by semitheoretical considerations and by computer calculations, that the resulting concentration profile of macrosolute is almost identical with that obtainable with a rectangular zone coextensive with the triangular one and carrying the same total mass. Next, practical procedures are described for the convectionless layering of very small triangular zones (50 microL or less). The linearity and stability of the zones are experimentally tested and verified. Finally, the method is applied to cartilage proteoglycan preparations that included either the monomeric molecules only or both the monomeric and the aggregated ones. The zonal results are compared with those obtained by using conventional boundary sedimentation. The two sets of results are seen to coincide fairly well, thus proving that the present technique can add to preparative zonal centrifugation the analytical precision of boundary sedimentation. A multimodal polydisperse system is suggested to describe the aggregated proteoglycan macromolecules.