Isopycnic Sucrose Gradient Centrifugation Research Articles

Subcellular Distribution of Proteins Synthesized in Slices of Rat Cerebral Cortex

Abstract The incorporation of l-[4,5-3H]leucine into slices of rat cerebral cortex has been measured, with emphasis on the time course of the distribution of label in subcellular fractions prepared by zonal isopycnic centrifugation in continuous sucrose gradients. Rapidly labeled proteins are found in both the soluble and membranous components of the nerve end particles. Except for a minor part of the mitochondrial protein, the synthesis of all membrane-bound proteins is inhibited drastically by cycloheximide. When synthesis is blocked by cycloheximide, the supply of label continues for some minutes to certain membrane fractions derived from synaptic structures, apparently by a rapid transport of newly synthesized proteins from cytoplasmic ribosomes in the neuronal perikaryon. An increased extracellular concentration of potassium ion inhibits and ouabain accelerates protein synthesis in some fractions. These effects suggest a coupling of the protein synthesizing system with the (Na+ + K+)-activated ouabain-sensitive adenosine triphosphatase in the plasma membranes.

Reactions at the 3′ Terminus of Transfer Ribonucleic Acid: V. SUBCELLULAR LOCALIZATION AND EVIDENCE FOR A MITOCHONDRIAL TRANSFER RIBONUCLEIC ACID NUCLEOTIDYLTRANSFERASE

Abstract The subcellular localization in rat liver of tRNA nucleotidyltransferase has been examined. The major portion of the activity in untreated subcellular fractions was present in the high speed supernatant fraction. Treatment of the liver homogenate with Triton X-100 or subjecting it to freezing and thawing increased tRNA nucleotidyltransferase activity about 50 to 70% suggesting that a substantial portion of the enzyme was present within organelles and unavailable for assay. Extraction of the various subcellular fractions revealed that mitochondria contained tRNA nucleotidyltransferase amounting to about one-third of the total activity in liver. Microsomes and nuclei were essentially devoid of activity. The activity in the mitochondrial fraction was shown not to be due to bacterial contamination. The distribution of tRNA nucleotidyltransferase upon isopycnic centrifugation in sucrose gradients was identical with the mitochondrial marker enzyme, cytochrome oxidase. Fractionation of mitochondria by digitonin treatment indicated that the enzyme was localized in the mitochondrial matrix. The catalytic properties of the tRNA nucleotidyltransferase activity in mitochondrial extracts were very similar to those of the homogeneous enzymes described previously in that it had an unusually alkaline pH optimum and was active with either ATP, CTP, or UTP. The enzyme required tRNA for activity and was essentially inactive with rRNA or in the absence of added RNA. These results suggest that mitochondria may contain a system for the maturation of tRNA molecules.

Structural components of Uukuniemi virus, a noncubical tick-borne arbovirus

Uukuniemi virus, the prototype of a new serological group of arboviruses, was grown in BHK21 cells and purified by isopycnic centrifugation in sucrose gradients. The purified virus had a sedimentation constant of about 450 S. The buoyant density of the virus was 1.20 g/cm 3 in CsCl and 1.18 g/cm 3 in sucrose. Sucrose gradient centrifugation of SDS-treated virus gave two RNA peaks (21 and 27 S). Two polypeptides of molecular weight 65,000–75,000 and about 25,000 could be separated from whole virus by SDS-disc electrophoresis. Two fractions were obtained by CsCl gradient centrifugation of Nonidet-treated virus. A fraction with a density of less than 1.25 g/cm 3 contained phospholipids and the larger protein (molecular weight 65,000–75,000). This fraction apparently contains disrupted virus envelopes. The other fraction with a density of 1.31 g/cm 3 contained RNA and the smaller protein (molecular weight 25,000). Electron micrographs of the latter fraction showed strands about 2.5 nm thick and over 1000 nm long, apparently uncoiled nucleoprotein of the virus. The mode of packing of the nucleoprotein within the virion is still obscure.

A survey of plants for leaf peroxisomes.

Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 m sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 m sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 m sucrose. In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ranged from 0.5 to 1.5 mumoles x min(-1) x g(-1) wet weight or a specific activity of 0.02 to 0.05 mumole x min(-1) x mg(-1) protein. Glyoxylate reductase activity was comparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 mumoles x min(-1) x g(-1) wet weight or 90 to 300 mumoles x min(-1) x mg(-1) protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial. Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50% of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 mumole x min(-1) x mg(-1) protein; for catalase. 8000 mumoles x min(-1) x mg(-1) protein, and for malate dehydrogenase, 40 mumoles x min(-1) x mg(-1) protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 mumole of glycolate oxidase x min(-1) x g(-1) tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane. Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses about the mechanism of photorespiration. Most of the catalase and part of the malate dehydrogenase activity was located in the peroxisomes. Contrary to previous reports, the chloroplast fractions from plants with photo-respiration did not contain a concentration of these 2 enzymes, after removal of peroxisomes by isopycnic sucrose gradient centrifugation.

FORMATION OF MITOCHONDRIA IN NEUROSPORA CRASSA. A STUDY BASED ON MITOCHONDRIAL DENSITY CHANGES.

The choline concentration used in the growth medium influences the density of mitochondria produced by the chol-1 mutant of Neurospora. Isopycnic centrifugation in sucrose gradients can be used to determine the density of mitochondria, and can resolve into two populations, mitochondria derived from a mixture of cells grown at low (1 microg/ml choline chloride) and high (10 microg/ml choline chloride) choline levels. In an experiment in which cells are shifted from low to high choline growth conditions, mitochondria obtained after varying time periods show a gradual decrease in density tending toward the level typical of high choline mitochondria. Over a 90-minute period of observation, during which time there is an increase of mitochondrial protein mass of approximately 50 per cent over that initially present, the mitochondria change density as a single population. These results are consistent with the view that mitochondria grow by random accretion of new lecithin into existing mitochondrial structures, and also that the mitochondrial population increases by division.

THE INFLUENCE OF PRECURSOR POOL SIZE ON MITOCHONDRIAL COMPOSITION IN NEUROSPORA CRASSA.

The chemical composition of mitochondria obtained from exponentially growing Neurospora can be varied by addition of choline or amino acids to the culture medium. The variation affects the phospholipid to protein ratio, and the density of mitochondria as determined by isopycnic centrifugation in sucrose gradients. These variations have been observed in biochemical mutant strains as well as wild type cultures. In a choline-requiring strain, two levels of choline supplementation to the medium have been defined: a low choline concentration just adequate to support maximal logarithmic growth, and a high choline concentration which permits maximal incorporation of radioactive choline into cellular lipids. Mitochondria isolated from cultures growing at the low choline concentration have one-half the phospholipid to protein ratio of those from high choline cultures, and their density is significantly higher. Artificial mixtures of the two types of mitochondria can be resolved into two populations by isopycnic centrifugation. The concentration of cytochromes (measured by mitochondrial difference spectra) and of malate and succinate dehydrogenases (measured by enzyme activity) were the same in both types of mitochondria, on a protein basis. The results suggest that during growth of the mitochondrial mass, the incorporation of phospholipid and protein components can vary independently. Direct kinetic measurements did indeed show that choline, added to a culture growing at low choline concentration, was incorporated into mitochondrial lipids at a rate faster than the incorporation of protein. The mitochondrial phospholipid to protein ratio can also be influenced by the level of leucine supplementation to a leucine-requiring mutant, so that with leucine concentrations above those required for maximal exponential growth, mitochondria of increasing density and decreasing phospholipid to protein ratio are produced. Additions of choline or amino acids to the minimal medium of wild type cultures influence mitochondrial composition in a manner directly comparable to that observed in biochemical mutant strains. The results suggest that mitochondrial composition, in general, is determined by rates of incorporation of the two major components, phospholipid and protein; that these rates can vary independently in response to precursor concentration in the culture medium; and that they normally operate at a precursor (substrate) concentration below saturation level.

THE PARTICULATE HYDROLASES OF MACROPHAGES. I. COMPARATIVE ENZYMOLOGY, ISOLATION, AND PROPERTIES.

The contents of selected hydrolytic enzymes of oil-induced peritoneal, normal alveolar, and BCG-induced alveolar macrophages have been studied. On a per cell or nitrogen basis the normal alveolar cells contained considerably more acid phosphatase, cathepsin, acid ribonuclease, lysozyme, and lipase than peritoneal cells. The BCG-induced alveolar macrophage exhibited increased levels of acid phosphatase, lysozyme, and lipase as compared to alveolar macrophages from unstimulated rabbits. The morphological differences between these cells was discussed and electron micrographs of the BCG-induced macrophage presented. Fractionation of the BCG-induced macrophage by differential centrifugation showed that 60 to 80 per cent of the total cell content of acid phosphatase, cathepsin, beta glucuronidase, acid ribonuclease, acid deoxyribonuclease, aryl sulfatase, lysozyme, and lipase were localized in a postnuclear fraction which sedimented at 15,000 g. This fraction also contained the majority of the mitochondria as evidenced by its content of cytochrome oxidase. Non-specific esterase was not localized to this fraction. A separation of the hydrolase-containing particles and mitochondria was achieved by isopycnic sucrose gradient centrifugation. Under the conditions employed, the mitochondria distributed at densities of 1.19 to 1.20, whereas the hydrolase particles sedimented to a density of 1.26 to 1.27. Each of the hydrolases including acid phosphatase, beta glucuronidase, cathepsin, lysozyme, and acid ribonuclease exhibited maximum activities in the same gradient fraction. The isolated granules exhibited enzymatic latency, and activation could be achieved by cycles of freezing and thawing or surface active agents. The majority of each of the hydrolytic enzymes could be liberated in a non-particulate form by mechanical trauma. Macrophages which had been stained supravitally with neutral red were fractionated by differential and gradient centrifugation. More than 70 per cent of the dye could be recovered in the particulate hydrolase fraction. The isolated, stained granules resembled those seen in the intact cell.