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S A Berkowitz

Publications and source records attributed to S A Berkowitz.

18 recordsLinked to original sources
PubMed1989

Protein purification by multidimensional liquid chromatography.

Recently Giddings discussed the prospect of combining two separation mechanisms in such a way that when "a sample is subjected to two displacement processes oriented at right angles to one another" a two-dimensional separation is carried out. In this review I have focused attention on the various ramifications of this concept in terms of combining two or more chromatographic techniques on-line to conduct MDLC for the purpose of purifying proteins. In general, the MDLC approaches discussed here were classified into two major categories. The first category involves the placement of several separation mechanisms in the same chromatographic work space (the chromatographic column). In this case the displacement processes are collinear. It is hoped that these new chromatographic packings and columns will display surface characteristics capable of a wide range of highly discriminating selectivities that can be modulated by mobile-phase changes to a greater extent than the nonspecific chromatographic techniques such as IEC and HIC. The ability to modulate the mobile phase to generate new selectivities is important in expanding the usefulness of these packings in comparison to the very high selectivity of affinity chromatography, which usually has little use outside its initial intended purpose to purify a particular protein. The second category involves the on-line physical coupling of two or more chromatography columns, each packed with a different chromatography material. Again the idea is to create or design a simple self-contained system that is capable of generating a wide range of high selectivities. Indeed, the on-line coupling of two different chromatographic packings for the purification of a single protein represents a line trace through "a discrete independent 2-dimensional system". These systems are highly attractive in large-scale purification, especially when using "on-off" chromatography, which eliminates the need for sophisticated gradient elution hardware. The purification of a single protein from its biological matrix is usually a multidimensional process utilizing several different separation technologies. By nature this leads to lengthy purifications that are frequently labor-intensive and expensive to scale up. It is my belief that future developments in the concept of on-line MDLC techniques involving complex chromatographic materials and column coupling will merge to create significant improvements in the protein purification process.

Chemical Phenomena↗
PubMed1987

Linear multidimensional liquid chromatography in the preparative scale purification of calmodulin from brain extract.

Rapid preparative scale purification of calmodulin from crude bovine brain extract is achieved in a single chromatographic run by physically coupling two different liquid chromatography columns which employ different separation mechanisms. In this case columns packed with newly commercialized 40-microns silica-based hydrophobic interaction and 5-microns micron silica-based weak anion-exchange chromatography media were used. The only sample preparation required for conducting this purification procedure is the addition of salt to the crude brain supernatant to promote the initial binding of calmodulin to the hydrophobic interaction chromatography media. Chromatography carried out on such linear arrangements of columns has been referred to as linear multidimensional liquid chromatography.

Animals↗
PubMed1981

Intrinsic calcium sensitivity of tubulin polymerization. The contributions of temperature, tubulin concentration, and associated proteins.

The calcium concentration required to inhibit tubulin polymerization by 50% (Ca2+ sensitivity) extends from the micromolar to the millimolar range and is a function of a number of factors that include 1) a steep, inverse dependence on tubulin concentration: two-cycle tubulin has lower Ca2+ sensitivity than pure tubulin (prepared by a novel method described under "Appendix"); 2) temperature: Ca2+ sensitivity shows a steep increase below 24 degrees C; 3) microtubule seeds: these decrease sensitivity to Ca2+ inhibition; 4) the presence of 16 S oligomers or microtubule-associated proteins. Ca2+ increases the critical concentration for microtubule protein and decreases the initial rate of polymerization. All tubulin preparations examined contain small amounts of calmodulin. However, the molar ratio of calmodulin to tubulin is less than 0.01, hence this protein is not required for high Ca2+ sensitivity. Nevertheless, calmodulin at high molar ratios can increase the sensitivity of microtubule assembly toward Ca2+. We conclude that tubulin possesses high intrinsic as well as a calmodulin-mediated Ca2+ sensitivity, and propose that high Ca2+ sensitivity may be a property of the nucleation process.

Animals↗
PubMed1981

Spurious protein activators of Bordetella pertussis adenylate cyclase.

A variety of proteins and tissue preparations (rabbit erythrocyte lysate, catalase, peroxidase, creatine phosphokinase, and lima bean trypsin inhibitor) contain protein activator(s) of the extracellular adenylate cyclase of intact Bordetella pertussis organisms. Stimulation of adenylate cyclase activity of up to 1000-fold over basal activity can be obtained. Activation of the adenylate cyclase is due to the presence of calmodulin in these protein preparations. The criteria to establish this were: Ca2+ dependence of the activation, inhibition by trifluoperazine, heat stability of the activator, chromatographic behavior like authentic calmodulin, and stimulation of cyclic nucleotide phosphodiesterase by the activators. The great sensitivity of the B.pertussis adenylate cyclase assay makes this and ideal system for the detection of trace amounts of calmodulin, in the presence of large amounts of other proteins.

Adenylyl Cyclases↗
PubMed1980

Turbidity measurements in an analytical ultracentrifuge. Determinations of mass per length for filamentous viruses fd, Xf, and Pf3.

An analytical ultracentrifuge has been used to measure light-scattering intensities by the transmittance method. The technique, which is applicable to particles of many sizes and shapes, has the principal advantage that samples can be kept free of dust during the measurements. Also, sample volumes are small, and the scanner and interference optics can be used simultaneously to obtain, for a given sedimenting boundary, turbidity steps at different wavelengths and the concentration step. In the present application the data yield mass per length estimates for three filamentous viruses, 19 100 daltons/nm for fd, 19 600 daltons/nm for Pf3, and 19 100 daltons/nm for Xf.

Coliphages↗
PubMed1980

Nucleation and the kinetics of microtubule assembly.

A kinetic model was developed for the purpose of interpreting scanning calorimetric data of microtubule assembly. The model consists of two steps. The first step is a highly exothermic nucleation with a strong dependence upon temperature and concentration. The second step is the elongation of the nuclei or microtubules by the addition of tubulin dimer to growing ends. Computer fits to the data provided the values of the parameters of the model. The model successfully simulated various experiments.

Calorimetry↗
PubMed1980

Calmodulin activates prokaryotic adenylate cyclase.

The adenylate cyclase of Bordetella pertussis is stimulated 100- to 1000-fold in a dose-dependent manner by calf brain calmodulin. The system has the following properties. (i) The activation is prevented by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and restored by Ca2+. (ii) Oxidation of the methionine residues of calmodulin abolishes the ability to activate the cyclase. (iii) Trifluoperazine inhibits calmodulin-activated cyclase. (iv) A troponin C preparation stimulates the B. pertussis cyclase with < 0.01 the potency of calmodulin. Although calmodulin has not been demonstrated in prokaryotes, this is an example of a (eukaryotic) calmodulin effect in a prokaryote.

Adenylyl Cyclases↗
PubMed1980

Observation of an exothermic process associated with the in vitro polymerization of brain tubulin.

The polymerization of tubulin has been studied with a high-sensitivity differential scanning microcalorimeter, with results which indicate that microtubule assembly can proceed via one or possibly two exothermic reactions. The amount of heat evolution has been found to be far in excess of GTP hydrolysis. The heat liberated has been observed to depend strongly upon the exact experimental conditions, varying from many hundreds of kilocalories per mole of tubulin dimer when dilute tubulin solutions are heated rapidly to a few kilocalories per mole of tubulin dimer when concentrated tubulin solutions are heated slowly. The results are tentatively interpreted in terms of the existence of at least two pathways for the formation of energetically distinct polymers. These findings indicate the importance of kinetic factors in studying tubulin polymerization.

Animals↗
PubMed1977

Separation and characterization of microtubule proteins from calf brain.

Electrophoresis of microtubule preparations purified from calf brain by repeated cycles of assembly and disassembly shows that they contain many proteins in addition to alpha- and beta-tubulin. These additional proteins constitute about 17% of the total material present after five cycles of assembly and disassembly. Both one-dimensional and two-dimensional (P.H. O'Farrell (1975), J. Biol. Chem. 250, 4007) electrophoretic techniques have been used to characterize them. They can be divided into two groups: one that contains proteins which remain in constant quantitative ratio to tubulin during the purification cycles, and one composed of proteins which are removed during purification, although inefficiently. Gel-filtration chromatography of cold-depolymerized microtubule preparations yields a polydisperse fraction of high molecular weight containing most of the non-tubulin proteins. This fraction contains flexible filaments about 100 A in diameter similar to those reported by R.A.B. Keats and R.H. Hall ((1975), Nature (London) 247, 418). It is suggested that these fibers are neurofilaments, and that they may be the major source of the group of inefficiently removed proteins.

Animals↗