Isoelectric focusing of brain adenylate cyclase

Mouse brain adenylate cyclase has been solubilized with Lubrol PX and separated by isoelectric focusing on polyacrylamide gels. The enzyme activity has been measured with a sensitive assay isolating cyclic AMP from Dowex and alumina columns. The technique allows a one-step analysis of this membrane enzyme from a heterogeneous sample within 6 hr.     

Calcium disodium edetate enhances type A monoamine oxidase activity in monkey brain

The effects of metal chelators on monoamine oxidase (MAO) isozymes, MAO-A and MAO-B, in monkey brain mitochondria were investigated in vitro. MAO-A activity increased to about 40% with 0.1 μM calcium disodium edetate (CaNa₂EDTA) using serotonin as a substrate, and this activation was proportional to the concentration of CaNa₂EDTA. On the other hand, MAO-A activities were decreased gradually with an increasing concentration of o-phenanthroline and diethyldithiocarbamic acid, but these metal chelators had no effect on MAO-B activity in monkey brain. The activation of MAO-A activity by CaNa₂EDTA was reversible. CaNa₂EDTA did not activate both MAO-A and MAO-B activities in rat brain mitochondria. Zn and Fe ions were found in the mitochondria of monkey brain. Zn ions potently inhibited MAO-A activity, but Fe ions did not inhibit either MAO-A or MAO-B activity in monkey brain mitochondria. These results indicate that the activating action of CaNa₂EDTA on MAO-A was the result of the chelating of Zn ions contained in mitochondria by CaNa₂EDTA. These results also indicate the possibility that Zn ions may regulate physiologically the level of serotonin and norepinephrine content in brain by inhibiting a MAO-A activity.     

Isoelectric analysis of 3H-pargyline-labelled monoamine oxidase in rat and carp

1. After selective binding of [3H]pargyline to either monoamine oxidase (MAO) A or MAO B in the rat liver, MAO B alone in the rat brain and MAO in carp brain and liver, molecular weight and isoelectric points (pI) of these MAO were determined by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and isoelectric focusing and results obtained were compared. 2. For all tissues tested, SDS-polyacrylamide gel electrophoresis of [3H]pargyline-bound samples revealed a labelled protein band of an apparent mol. wt of 60,000 da. 3. Estimation of radioactivity of [3H]pargyline bound after isoelectric focusing revealed a single protein band with acidic pI values of about 5.5 for rat brain and liver MAO B. 4. Moreover, the pI values of about 7.5 were obtained for carp brain and liver MAO. This basic value was also found for MAO A in the rat liver MAO A.     

Isoelectric focusing of cytochrome P450: isolation of six phenobarbital-inducible rat liver microsomal isoenzymes

A procedure for the isolation of native proteins from membranes by isoelectric focusing is described. It was used to resolve into six components the major fraction of cytochrome P450, obtained from liver microsomes of phenobarbital-treated rats, after chromatography on DE-52 cellulose. When eluted from the gel, these proteins are in a native form as shown by (a) the light absorption spectra of the Soret region of their reduced carbonyl derivatives, all characterized by maxima around 450 nm, and (b) their enzymatic activities toward three different substrates. Characterization by a monoclonal antibody and partial sequence analysis of tryptic peptides reveal that three of the IEF-purified proteins have P450IIB1 character, whereas the other three are related to P450IIB2.     

Titration of human brain type-B monoamine oxidase

The interaction of the substrate-selective irreversible inhibitor J-508 [N-methyl-N-propargyl-(1-indanyl)-ammonium hydrochloride] with the B form of human brain monoamine oxidase has been investigated, and the conditions necessary for this inhibitor to titrate the concentration of this enzyme form determined. It was found that the concentration of monoamine oxidase-B determined in this way was the same when either benzylamine or -phenethylamine was used to assay for activity, which would indicate that this enzyme form is not heterogeneous. Furthermore, the variation in activity from sample to sample was found to be due to a variation in the concentration of available monoamine oxidase-B active centers, rather than due to a variation in the molecular turnover numbers of this enzyme form towards its amine substrates.     

Isoelectric focusing in polyacrylamide-agarose

A procedure using a 2.5% acrylamide-0.5% agarose gel for slab or tube isoelectric focusing is described. This composite gel is durable and enables a rapid focusing of high-molecular-weight compounds.     

Isoelectric focusing studies of bacteriorhodopsin

Purified bacteriorhodopsin (BR) samples show a minimum of four isoelectric forms in immobilized pH gradient isoelectric focusing gels. The bands occur as doublets with isoelectric points (pI) centered at 5.20 (principal species) and 5.60. In typical preparations additional bands may be observed at 4.90, 5.07 and 5.50. Purple membrane (PM) was proteolyzed with papain to calibrate the pI shift produced by changing the number of charges on the protein. Asp-242 is removed during the first cleavage between residues 239 and 240 resulting in the loss of a single negative charge and a shift of the principal doublet by +0.35 pH units to pI 5.55. The second papain cleavage occurs between residues 231 and 232 which removes Glu-232, -234 and -237 and shifts the pI by +0.60 pH units to pI 6.10. The +0.60 pH shift upon the second papain cleavage is consistent with the loss of two negative charges and is supported by prior evidence that at least one of the three glutamate residues lost during the second proteolysis step is protonated and neutral in the intact protein. The native and proteolyzed products of BR retain the characteristic 550 nm absorption maxima for solubilized BR. A model for the structural origin of the pI heterogeneity of BR species in proteolyzed PM is presented.     

High resolution of peroxidase-indoleacetic Acid oxidase isoenzymes from horseradish by isoelectric focusing

Several improved techniques for isoelectric focusing of isoenzymes in polyacrylamide gel slabs were developed. Using these techniques, three commercial sources of horseradish peroxidase were each examined with three commercial sources of carrier ampholytes to determine their respective isoenzyme profiles.     

Isoelectric focusing of cassava protoplasts

Cassava (Manihot esculenta Crantz) protoplast was analyzed by using isoelectric focusing techniques. Two populations, representing 68 and 32% of the total sample, with mean isoelectric points of 4.48 and 4.60, were obtained using mesophyll protoplasts. The use of this technique allows demonstration of a discontinuous distribution of protoplast isoelectric point from one species according to their surface potential.     

Isoelectric focusing in Pevikon slabs

A mixture of Pevikon C-870 and Sephadex G-75 superfine is proposed for isoelectric focusing in granular beds. The beds are easy to prepare, and pH gradients are stable for 36 hr under the given conditions. Precise and clean fractionations are obtained with excellent recoveries of focused substances.     

Isoelectric focusing electrophoresis of lignin.

Isoelectric focusing is introduced as a technique for the analysis of macromolecular lignin. The analysis is performed in a pH gradient from 3.5 to 10. Separated lignin fragments are visualized under uv light or by silver staining. The method can be used to distinguish between differently processed lignin preparations and to identify their components. Even the slight modification resulting from attack by ligninolytic enzymes could be detected.     

Isoelectric focusing of myelin membrane proteins

Human myelin was isolated from the white matter of autopsy brains. Myelin proteins were characterized by isoelectric focusing in ultrathin slab gels in a pH range from 3.5 to 10 after solubilization with urea and Nonidet P 40. The protein profile in the acidic region (pH below 6.2) revealed at least twelve faint bands which comprised only a few percent of the total myelin proteins. Most of the myelin proteins were focused in the neutral range (pH 6.2–7.8) which showed two sharper and three broader major bands, the total number of bands in this region being about twenty. The basic pH range (pH above 7.8) contained about 30% of the proteins, and revealed a very intense band near the cathode with seven to nine weaker bands below pH 9.0. When the myelin was partially delipidated prior to solubilization, an additional broad band was observed at the area pH 8.0–8.5.