John Graham

Nycodenz: A new nonionic iodinated gradient medium

Abstract The properties of Nycodenz, a new nonionic iodinated density gradient medium, are described. This compound shares a number of characteristics with its related compound metrizamide. However, the stabler, more inert nature of Nycodenz endows it with a number of favorable properties as a gradient medium.

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

SummaryLeakage of ions (Na+, K+) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by ‘pore-forming’ agent has been studied. Leakage from intact cells isinduced by protons and by divalent cations such as Cu2+, Cd2+ or Zn2+. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented by low concentrations of the same cations and by higher concentrations of Ca2+, Mn2+ or Ba2+; Mg2+, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca2+) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na+, K+, Rb+, NH4+) and divalent (Mg2+, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn2+ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.

Isolation of the major subcellular organelles from mouse liver using nycodenz gradients without the use of an ultracentrifuge

Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analysis.

A rapid method for the separation of large and small thymocytes from rats and mice

Thymocytes from rats and mice have been separated into large and small cell populations in high yield and purity by isopycnic centrifugation in a discontinuous (three-layered) gradient of Percoll. Cells are mixed with the middle layer and during low-speed centrifugation the small, denser cells sediment to the bottom interface whilst the large, less dense cells float to the top interface. Red blood cells, and small debris particles separate into the bottom and top layers respectively. Studies on the uptake of [3H]TdR show that the small cells are non-dividing while more than half of the large cells are capable of division.

Buoyant densities of macromolecules, macromolecular complexes, and cell organelles in Nycodenz gradients

Nycodenz is a new nonionic iodinated density gradient medium which has several advantages over metrizamide. Although, overall, biological samples band at similar densities in Nycodenz and metrizamide gradients, a number of significant differences were found. As compared with metrizamide, not only does Nycodenz appear to interact less with proteins but also the buoyant density of chromatin is less affected by the amount loaded onto the gradient. A high degree of resolution is obtainable using Nycodenz gradients; thus, it is possible to separate density-labeled DNA and to subfractionate subcellular membrane fractions.

A new, rapid, one-step method for the isolation of platelets from human blood

We describe a new, rapid method for the isolation of platelets from human blood using a single centrifugation step through Nycodenz, an inert, nontoxic, nonionic medium. As judged by aggregation and nucleotide release studies, the platelets are recovered in high yield and in excellent functional condition.

Localization of vasopressin in synaptic vesicles of extra-hypothalamic rat brain

The subcellular localization of vasopressin (VP) from extra-hypothalamic areas of rat brain was investigated by measuring its distribution (a) along a continuous sucrose gradient; (b) during the preparation of isolated nerve endings (synaptosomes) and (c) during the preparation of synaptic vesicles. Quite large amounts of vasopressin are isolated in the same fractions as mitochondria, as well as synaptosomes. Osmotic rupture of membrane bound organelles in the homogenate results in the vasopressin being measured largely in the fraction containing synaptic vesicles. These results would suggest that vasopressin could be released by nerve terminals which is consistent with the hypothesis that it may have a neurotransmitter/neuromodulator function in the CNS.

Isolation of platelets from human blood by free-flow electrophoresis

We are using platelets as a model system in an investigation into the alterations in the calcium-stimulated ATPase which occur in primary hypertension in man. In order to make valid comparisons between platelets prepared from normal subjects and from hypertensive patients it is essential that the purity of the platelet preparation be high and reproducible. Most clinical studies on the human platelet require only 20-30 ml blood and low-speed differential centrifugation is very successful in obtaining acceptable recoveries from such samples. On the other hand many biochemical studies are carried out on platelet+ch plasma derived from one or more units of donated blood: in these cases high recoveries are relatively unimportant. For our own studies we needed to maximise the recovery of platelets from 150-200 ml of blood. Purification by differential centrifugation required multiple cycles of sedimentation, resuspension and washing which was both timeconsuming and led to progressive platelet disruption. Preliminary to our biochemical studies therefore we have investigated methods of harvesting platelets from human blood. A new method is presented in this paper which uses free-flow electrophoresis. This technique involves injection of a buffy-coat fraction into a vertically moving column of buffer across which a potential difference is imposed. Because of the lower surface charge density on the platelets compared to that on erythrocytes or white cells, the platelets can be resolved from these other blood cell elements. The functional integrity of the platelet preparation has been assessed by measurement of nucleotide levels in the cells before and after stimulation by a variety of agents which cause aggregation.

Receptor-mediated endocytosis of enterokinase by rat liver: Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30-45 kDa range.

Fractionations in Zonal Rotors

Publisher Summary This chapter discusses the fractionations in zonal rotors. A typical zonal rotor is in the form of a closed cylinder containing a central core and four or more radial vanes or septa, which end close to the wall of the rotor, effectively dividing its interior into four or more sectors. In a spinning rotor, the centrifugal field is radial; thus, only that part of the centrifugal field passing through the centre of a tube in a swing-out rotor is parallel to the wall of the tube. Only the particles at the centre of the sample zone will sediment directly towards the bottom of the tube; all other particles will be deflected toward the wall of the tube to an extent proportional to their distance from the centre of the sample zone. This wall effect leads to a loss of resolution that does not happen in the sector-shaped compartments of a zonal rotor. To minimize disturbance of the gradient and to maintain sharp sample zones, these rotors have been designed to be loaded and unloaded while rotating, that is, dynamically, so that the perturbation and reorientation of a gradient, which occurs during acceleration and deceleration of swing-out and fixed-angle rotors, are eliminated.