Norman G. Anderson

Analytical differential centrifugation: An analysis of the sedimentation properties of synaptosomes, mitochondria and lysosomes from rat brain homogenates

Abstract A newly developed analytical differential centrifugation procedure for use in angle rotors was used to analyze the sedimentation of certain brain subcellular particles. Particle sedimentation was measured over a wide range of applied forces, and a curve descriptive of this sedimentation was computed. Precise conditions for complete and fractional sedimentation of mitochondria, choline acetyltransferase-containing nerve-end particles, and lysosomal activities from rat brain cerebral cortex homogenates were established. Mitochondria, nerve-end particles, and lysosomal activities began to sediment together, but all mitochondrial cytochrome oxidase sedimented before nerve-end particles and lysosomes. Even though populations overlapped, a discrete sizing of mitochondria and nerve-end particles was accomplished. Mitochondria sedimentation was similar to an ideal particle even though brain mitochondria are composed of at least two populations. These data are compared with those used by other investigators to provide a basis for making a decision on significant differences among diversified separation conditions.

Analytical techniques for cell fractions: XIV. Use of drainage syphons in a fast-analyzer cuvet-rotor

Abstract A cuvet-rotor, designated G-IIB, has been developed that contains small-bore syphons attached to each of the fifteen cuvets. Drainage of reactants from the transfer disc was found to be approximately 90% complete in 1 second and 99% complete after 2.5 seconds during acceleration to 1000 rpm. Mixing of protein-biuret reagent solution and 30% sucrose occurred in 1 second when a vacuum was used to pull air back through the syphons and cuvets. Drainage of the cuvets through the syphons was almost quantitative (>99%) when air pressure and one cycle from 400 rpm to 1000 rpm and back to 400 rpm was used. The interval between successive sets of analyses may therefore be 1 minute or less. A method of heat cycling to make the Telflon cuvet-spacer conform to the glass windows is presented.

The development of automated systems for clinical and research use

Abstract Basic research necessary for the development of advanced methods for clinical chemistry are reviewed. It is concluded that a new class of fast analyzers are required which are inherently adapted to computer data reduction. A new analytical system using centrifugal force to transfer and mix fluids in a multiple-cuvet rotor yields data signals at intervals of 3.3 milliseconds. It is applicable to a variety of analyses, but is especially suited to the determination of enzymes by measuring reaction rates.

Analytical techniques for cell fractions: XIX. The cyclum: An automatic system for cyclic chromatography

Abstract An automatic system, termed a Cyclum, is described which allows column chromatographic separations to be repeated precisely a large number of times. Provision is made for the adjustment during operation of parameters such as equilibration, wash, elution, and sample flow times and duration of fraction collection. The system is applicable to both analytical and preparative use in various types of column chromatography (e.g., affinity, gel filtration, ion-exchange), but has been especially developed for separations based on immunosorption.

Analytical techniques for cell fractions. XX. Cyclic affinity chromatography: principles and applications.

Abstract Principles and applications of an automatic cyclic affinity chromatography system, especially as applied to separation of proteins by immunosorption, are described. Use is made of columns of either immobilized antigens or antibodies to separate from a mixture, in a repetitive fashion, a desired protein or proteins by immunoadsorption followed by elution or to take from a mixture all proteins but the desired ones by allowing these latter to go through unadsorbed. The amplification provided by the cyclic use of the system and the biological amplification of hyperimmunization in achieving useful yields of desired proteins is discussed. Experience in the use of eluting solutions which do not cause damage to either the fixed or eluted proteins is presented. Representative separations done with the system are described, illustrating the different modes of use.

Analytical techniques for cell fractions: XV. Rotor B-XXIX—A new high-resolution zonal centrifuge rotor for virus isolation and cell fractionation

Zonal rotors previously developed in the MAN Program have found wide use in the isolation of subcellular particles, viruses, proteins, and nucleic acids (1,2). Separations may be made on the basis of sedimentation rate or banding density in gradients of large volume on a preparative scale with high resolution. In nearly all rotors previously described the gradients are recovered from the rotor by displacement using a dense solution pumped to the rotor edge (3). For a number of interesting separations, however, it is desirable also to be able to recover gradients or portions of the gradients from the rotor edge (4). Mitochondria, lysosomes, cell membranes, and peroxisomes have very similar banding or isopycnic densities in sucrose gradients but have different sedimentation rates. These particles start together from the sample zone, form rather well-resolved zones during rate sedimentation, and then bunch together in sucrose gradients in the region of 4045% w/w sucrose. Removal of the denser or edge portion of the gradient and its replacement with fresh solutions at the proper intervals should allow the recovery of particle zones not only having different sedimentation rates but also with a larger range of sedimentation coefficients. Edge-unloading rotors would also solve an important isolation

Tumor lipids: Characterization of the lipids isolated from membranous material

Abstract Membranous material was prepared from Ehrlich ascites carcinoma cells by zonal centrifugation. The distribution of fatty acids among the individual neutral lipid and phospholipid classes isolated from the membrane preparation was determined. Survey electron microscopy indicated that the preparations consisted of vesicles of rough surfaced endoplasmic reticulum, lysosomal-like structures, virus-like particles, few intact mitochondria, and unidentified amorphous material, in addition to plasma membranes. Lipids and proteins accounted for approximately one-third and two-thirds of the membranous material. Phospholipids accounted for two-thirds of the total lipids. The membranous material contained higher levels of free fatty acids, cholesterol, diacyl phosphatidyl choline, diacyl phosphatidyl ethanolamine, and lower levels of glyceryl ether diesters and cholesterol esters than whole cells. The fatty acid composition of all the lipid classes isolated from the membranous material, except phosphatidyl inositol and cholesterol esters, was similar to the fatty acid distribution of the corresponding classes obtained from whole cells. Cholesterol esters contained elevated levels of 16:1 and stearic acid accounted for 88% of phosphatidyl inositol fatty acids. Phosphatidyl choline and phosphatidyl ethanolamine isolated from the membranous preparations contained 1% or less alkyl acyl phosphatides, and plasmalogens were not detected. The lipids of whole Ehrlich ascites cells have been shown to contain high levels of ether-linked lipids, whereas these data indicate that alkyl and alk-1-enyl glyceryl ethers are virtually absent from membrane preparations of these cells. These results indicate that the ether-linked lipids are not distributed equally among all cell structures.

K-series centrifuges: III. Effect of core taper on particle capture efficiency

Abstract The taper in the K-II core was designed to aid in maintaining resolution during dynamic unloading. Static unloading has proved to be superior in practice however, thereby eliminating the requirement for a taper. Since, as shown here, superior recovery is obtained with a nontapered core, the latter design has been adopted for the K-III titanium continuous-sample-flow-with-banding rotor (17).

Analytical techniques for cell fractions X. high-pressure ninhydrin reaction system

Automated bioanalytical systems are usually scaled on the basis of available components since the initial objective is, generally, to prove underlying principles rather than to construct a system having optimum performance. As additional devices are developed, the problems of resolution, accuracy, sensitivity, size, and maintenance become of increasing interest. For both research purposes and clinical use, small reliable instruments are required. We are interested in exploring the factors that appear to be limiting in the development of one class of such instruments, namely, those depending on ion exchange. While attention in this laboratory has previously been centered on the development of automated systems for nucleotides and related compounds (l-3) and for sugars (4), our interest is now directed toward the problem of reducing the size and analysis time for the most widely used system: the amino acid analyzer of Spackman, Stein, and Moore (5), Piez and Morris (6)) and Hamilton (7). Theoretical (8) and experimental (7) studies on ion-exchange separation have indicated that a limiting factor is diffusion of the solutes to be separated into and out of the ion-exchange bead. Interest has therefore focussed on means for shortening the distance through which solutes have to diffuse, either by decreasing the volume of the ion-exchange bead (7) or by confining the exchange resin to the surface of an impermeable bead (9). The limiting resolution will probably be obtained by using both approaches simultaneously, which will result in high back pressures. Unfortunately, high-pressure pumps having accurate deliveries at very low flow rates are generally large and difficult to build and maintain,

Variable-speed drive for the mechanical stage of a microscope used as the optical component in a microdensitometer

Abstract An inexpensive yet versatile device for driving the mechanical stage of a microscope is described. The device provides for continuous variable-speed control and positively monitors the distance traversed at short and known increments of stage travel. This device was attached to the horizontal stage of a microscope used as the optical component of a microdensitometer in order to facilitate the accurate and precise determination of migration distances when scanning developed electrophoresis patterns.