Howard K. Schachman

Studies on the macromolecular organization of microbial cells

Five different methods of preparing extracts from bacterial cells yielded similar distributions of macromolecular constituents as indicated by ultracentrifugal patterns. Extracts from a number of different bacteria showed essentially similar patterns. Three principal components were found, with sedimentation constants of approximately 40, 29, and 5 S. In many extracts, it was evident that the most slowly moving peak consisted of two components, a broad diffuse boundary and a sharp spike containing desoxypentose nucleic acid. Extracts from the photosynthetic bacterium Rhodospirillum rubrum contained, in addition to the above-mentioned components, a component of very large size (sedimentation constant approximately 190 S), with which the entire pigment complement of the cells was associated. Chemical analyses of crudely fractionated extracts showed that the bulk of the pentose nucleic acid of the cells was associated with the particles of sedimentation constants from 20 to 40 S, while the desoxypentose nucleic acid was almost all present in the fraction of lower sedimentation rate.

The isolation and characterization of a macromolecular ribonucleoprotein from yeast

Abstract Studies have been conducted on the isolation and stability of large particles, with a sedimentation coefficient of 80 S , observed in extracts from yeast. With knowledge of some of the factors which cause the decomposition and aggregation of these particles, it was possible to obtain a solvent mixture in which the particles were stable. Isolation of the 80 S particles showed that they composed at least 1.5% of the wet weight of the cells and about 50% of the ribonucleic acid (RNA). Analyses indicated that the 80 S particles were a ribonucleoprotein containing about 42% RNA and 58% protein. Ultracentrifugation and electron microscopy showed that the particles were almost uniform in size with a diameter of about 21 mμ.

Ultracentrifugal analysis of dilute solutions

Abstract Absorption optics both in the visible and ultraviolet regions of the spectrum have been used in ultracentrifugal studies at concentrations of about 0.001%. The experimental techniques have been described, and methods of handling the photographic plates to determine sedimentation coefficients, apparent diffusion coefficients and distribution of sedimentation coefficients are illustrated in detail. Different types of experiments have been performed with preparations of a bacterial cytochrome, human hemoglobin and calf thymus deoxyribonucleic acid. In view of the risks that convective disturbances may invalidate ultracentrifuge experiments at very low concentrations, special attention was directed toward testing the reliability of the studies reported herein. It is shown that a cytochrome boundary behaved ideally and the apparent diffusion coefficient was independent of centrifugal field. Similarly the spreading of the DNA boundary gave a calculated distribution curve which was independent of time of sedimentation, speed of the rotor and of concentration of the material. The polydispersity was conclusively demonstrated by comparison of the patterns of a fractionated sample and the original preparation of DNA. Measurement of accurate sedimentation coefficients at 0.05 and 0.005% was illustrated with a preparation of carboxyhemoglobin. These studies taken together show the reliability of the measurements at very high dilutions. Other applications of absorption techniques in ultracentrifugation are discussed.

Molecular-weight determinations during the approach to sedimentation equilibrium.

Abstract Discussed herein are different methods for treating the data obtained in an ultracentrifugal study during the approach to sedimentation equilibrium. All of these methods are based on the original proposal of Archibald. Equations are presented and the advantages and disadvantages of the various procedures are illustrated with results from experiments on purified proteins and known mixtures of homogeneous materials. Since it is difficult to obtain reliable data at the bottom of existing ultracentrifuge cells, special efforts have been directed toward this problem. It was found that the addition of a small amount of a dense, inert, transparent liquid, such as silicone fluid, to the ultracentrifuge cell facilitates the determination of precise molecular weights at the bottom of the cell. In effect, this dense liquid creates in the ultracentrifuge cell a false bottom of the correct shape. Moreover, the transparency of this cell bottom greatly simplifies the detection of aggregated material in the solution. Precise values of the molecular weights of ribonuclease and β-lactoglobulin have been obtained, and the results are in excellent agreement with values derived by other methods. Furthermore, correct weight-average molecular weights have been determined for various known mixtures of these proteins. It was also found that inhomogeneity can be readily demonstrated. For certain types of mixtures the Archibald method gives not only accurate values of the weight-average molecular weight but it also provides a reasonable estimate of the molecular weight of the heavy component.

Ultracentrifuge studies with absorption optics: I. An automatic photoelectric scanning absorption system☆

Abstract An automatic photoelectric scanning system is described for use with absorption optics in the ultracentrifuge. The system consists of a constant-speed drive assembly which by means of a flexible, self-centering coupling causes a lead screw to rotate, thereby imparting linear motion to the photomultiplier-slit assembly. In 5 sec. the photomultiplier scans a magnified image of the ultracentrifuge cell. High resolution is achieved by the use of a slit about 25 μ in width in front of the photocathode. Appropriate electronic circuits based upon well-established data sampling techniques provide for simultaneous measurement of the intensity of light and its derivative or, alternatively, the optical density and its derivative as a function of distance in the cell. Direct measurement of light intensity furnishes a sensitive index for the proper alignment of the optical system; optical density measurements supply the necessary data for sedimentation velocity and sedimentation equilibrium experiments. Detailed consideration is given to tests of the performance of the instrument. Sedimentation velocity experiments with bushy stunt virus show that results of high accuracy are obtained readily. These data include not only the determination of sedimentation coefficients but also the concentration and its change with time during an experiment. Results of molecular weight determinations of adenosine by sedimentation equilibrium are also given. The precision of these measurements is high. Although the accuracy also seems to be high, certain reservations are discussed and further work is outlined which should lead to greater reliability. At its present state of development the automatic scanner represents a vast improvement over the photographic method in terms of versatility, convenience, and precision. As yet the full potential of the method, especially with regard to accuracy, has not been realized.

A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase.

The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod–Wyman–Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R–T equilibrium by using highly deuterated, 1H,13C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L′) between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is “invisible” in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R–T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R–T equilibrium, consistent with the Monod–Wyman–Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons.

Ultracentrifuge studies with absorption optics: 3. A split-beam photoelectric, scanning absorption system

Abstract An automatic split-beam scanning absorption system for the ultracentrifuge is described. The system consists of a four-speed drive assembly which controls the rate of movement of a photomultiplier across the magnified image of the ultracentrifuge cell. The photomultiplier housing contains a turret which permits the selection of any of six different slits in front of the photocathode. High resolution is achieved by the use of slits about 35 μ in width. Double sector cells are used in the rotor so that two light bursts strike the photomultiplier in quick succession followed by a long dark period while the rotor completes one revolution. The cell is filled in such a way that the first pulse after the long dark period corresponds to the solvent compartment and the second sector contains the solution of interest. A logarithmic circuit converts the photomultiplier voltages to a logarithmic scale. An electronic switching arrangement is incorporated which permits the separation of the light pulses and the routing of the two signals into two holding circuits, one for the reference cell and the other for the sample. The outputs of the holding circuits representing the logarithms of the light intensity through the two compartments of the cell are then fed to a difference amplifier where subtraction is achieved. The resulting signal is then directed to a recorder which plots automatically the optical density as a function of position in the cell. In this way the single beam passing through the vacuum chamber is split by the chopping action of the rotor to give two individual pulses which are then stored and compared. Experiments are presented to demonstrate that the split-beam system automatically compensates for nonuniform illumination resulting from inadequacies in the optical system itself. Performance tests are given to show the linearity of the recording system in terms of the optical density of the liquid in the sample cell. The switching system is shown to perform satisfactorily so that switching is accomplished in 1 μsecond thereby permitting the separation of the light pulses which occur within 6 μseconds when the rotor operates at 60,000 rpm. Various applications of the optical system are given. These include sedimentation coefficient determinations as well as the evaluations of concentrations and molecular weight determinations by the sedimentation equilibrium technique. In both types of experiments the results were in excellent agreement with known values. Other applications include the analyses of interacting systems both by transport and equilibrium methods and the measurement of small differences in sedimentation coefficients.

Ultracentrifuge studies with absorption optics: II. Incorporation of a monochromator and its application to the study of proteins and interacting systems

Abstract The adaptation of the ultracentrifuge for the incorporation of a monochromator into the absorption optical system is described. This involved raising the ultracentrifuge onto new, higher supports which also provide an adaptable, rigid mount for the monochromator. Remote control knobs and an illuminated panel facilitate the adjustment of the slit width and the wavelength of the light used in the photoelectric scanning absorption system. Procedures for aligning and focusing absorption optical systems are given and different tests are illustrated. These include techniques for determining the correct vertical height of the light source so as to give parallel light through the ultracentrifuge cell, the correct horizontal position of the light source in terms of the optic axis, the appropriate adjustment of the 45 ° mirror of the optical system, and the correct position of the camera lens for focusing on the midplane of the cell. Some of the spurious reflections which plague the absorption system are discussed along with appropriate remedies. The combined monochromator-scanning system is used for determining concentrations, sedimentation coefficients, and spectral properties of proteins. Finally, the optical system is used in both sedimentation velocity and sedimentation equilibrium experiments for the analysis of interactions between colorless macromolecules and small, colored ions and molecules. A sedimentation equilibrium technique of considerable promise for such studies is described and illustrated for a mixture of bovine plasma albumin and methyl orange.

Studies on the alkaline degradation of tobacco mosaic virus. I. Ultracentrifugal analysis.

Abstract The kinetics of the alkaline degradation of tobacco mosaic virus (TMV) has been followed by ultracentrifugal analysis and viscometry, and some of the products were characterized chemically and electrophoretically. The discrete, sharp boundaries observed in the ultracentrifuge indicate the existence of relatively homogeneous components of size intermediate between that of TMV and the smallest degradation product which has a molecular weight of the order of 105. The character of the reaction changes markedly between 0 and 25 °C., and different products were obtained at the two temperatures. Contrary to the interpretation of similar studies by other workers, it was shown that some of the intermediates and even some of the final products are the result not of degradation of larger components but rather of aggregation of smaller degradation products. Only two components of the many that were observed are apparently true degradation products. One of these is a protein with molecular weight of about 105 and the other is a nucleoprotein rodlike particle of length about 1 3 that of TMV. The free nucleic acid liberated during the degradation was not characterized. From these studies it is suggested that the virus possesses some structural features differentiating 1 3 of a given particle from the remaining 2 3 . A mechanism of degradation is proposed in which the protein units are stripped rapidly from one end of a virus particle until 2 3 of the particle is disrupted. In this way the relatively stable nucleoprotein particle, 1 3 the size of the virus, is presumably produced. Some particles with the sedimentation and electrophoretic characteristics of TMV are completely resistant to degradation under the same conditions which lead to the breakdown of the bulk of the virus.

Dissociation of hemoglobin into subunits: Monomer formation and the influence of ligands☆

Abstract The formation of monomer from several hemoglobins has been investigated by sedimentation equilibrium. The use of the split-beam photoelectric scanning absorption optical system has enabled observations to be made routinely down to 1 μg/ml. (6.2 × 10−8 m -heme) with strict spectral control of the integrity of the hemoglobin molecule. The results show that the dissociation constant of dimer to monomer at neutral pH and moderate ionic strength is so small that monomer is present in reversible equilibrium with dimer only in fractions too small to be detectable. Any appreciable monomer formation is irreversible and accompanied by usually pronounced spectral changes. This irreversible monomer formation is probably a consequence of the presence of heavy-metal ions in solution and may be inhibited by 10−3 m -EDTA. Hemoglobin ligands possessing chelating ability also inhibit monomer formation.