Enzyme electrophoretic analysis

Electrophoretic analysis reveals that there are more than 20 different polypeptides in the preparation [39,289,290]. The status of these proteins as enzyme subunits is not clear. However, some are associated with FeS centres and may be isolated in defined subcomplexes (see below). Some may constitute specific binding sites for ubiquinone [281]. No success in isolation of rotenone-sensitive Complex I with a smaller number of constituent polypeptides has been reported. 

Type III. D-Glucopyranosyl residues are transferred from uridine 5-(a-D-glucopyranosyl pyrophosphate) and D-glucosyluronic acid residues from uridine 5-(D-glucopyranosyluronic acid pyrophosphate) in a 1 1 ratio. The resulting polysaccharide was identified by precipitation with a specific antiserum, followed by hydrolysis with acid or with a depolymerase known to hydrolyze Type III polysaccharides only. The results of immuno-electrophoretic analysis showed that the enzymically synthesized polymer has a molecular weight of the same order of magnitude as that of the polysaccharide produced in vivo. ... 

The main acceptor for ADP-ribose in the in vitro labeling experiments was a protein of mol. wt. of 115—125 kD (Fig. 2). It was assumed to be poly(ADP-ribose) synthetase, since this enzyme has been found to catalyze self-modification reactions [18]. This assumption was verified by incubating nuclear matrices isolated from unlabeled cells with labeled NAD. Figure 4 shows that the tracer was indeed incorporated into acid-insoluble material in a time-dependent reaction. The fact that a specific inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide, almost completely inhibited the reaction, indicated that the reaction was catalyzed by this enzyme. This conclusion was also supported by electrophoretic analysis of the labeled products formed (not shown). 

The various catalytic properties of aconitase, for example, might be due to the presence of several proteins so similar in many respects that separation is difficult. Nevertheless, as we have already pointed out, the two catalytic properties of aconitase have been separated in A. niger. Furthermore, the claim that these various catalytic properties are due to a single protein because the preparation is declared pure on the basis of ultracentrifugal and electrophoretic analysis is unjustified, because these methods demonstrate only that the protein components of the material under study are similar in size and mobility. Most protein chemists would require more severe criteria of purity, such as end-group analyses. This line of reasoning applies a fortiori to the multiple-enzymes systems. 

An a-D-glucosidase purified from flint corn by precipitation with ammonium sulphate, ion-exchange chromatography, and gel-filtrations was homogeneous in ultracentrifugal and disc electrophoretic analysis. The enzyme (6.5 Sy mol. wt. 6.5 X 10 by gel filtration) showed a pH optimum of 3.6 for both maltose and soluble starch. The ratio of velocities of hydrolysis for maltose, phenyl a-D-glucopyranoside, and soluble starch was estimated to be 100 14.3 6.1. The a-o-glucosidase hydrolysed soluble starch by an exo action. 

The total number of polypeptides in the culture medium was 8-20 bands according to electrophoretic analysis (Figure 1). The protein export was inhibited by actinomycin D, cycloheximide, 2-deoxy-D-glucose and also by concanavalin A (Con A). In the presence of Con A (400 pg/ml), the enzymes were accumulated entirely inside the cells but not in the periplasm or the cell wall (Figure 2). At the same time the lectin had no influence upon cell growth and expression of invertase and acid phosphatase associated with the cell envelope. The activity accumulated inside Con A - treated cells was released into the culture medium on a-methylmannoside addition (Figure 3). The process was not affected by cycloheximide but was inhibited by sodium azide. The effect of Con A appears to be conditioned by its interaction with the plasma membrane of intact cells. Besides Con A, we detected peroxidase interaction with the plasmalemma of intact cells and alkaline phosphatase internment under physiological conditions. 

Wuilloud, R.G. et al.. The potential of inductively coupled plasma-mass spectrometric detection for capillary electrophoretic analysis of pesticides. Electrophoresis, 26,1598, 2005. Morozova, V.S., Levashova, A.I., and Eremin, S.A., Determination of pesticides by enzyme immunoassay, /. Anal. Chem., 60, 202, 2005. 

The sulfate is obtained by evaporating the aqueous layer in vacuo. The hydrochloride can be obtained in the same way but using HCl instead of H2SO4. SAM-HCl has a solubility of 10% in H2O. The salts are stable in the cold at pH 4-6 but decompose in alkaline media. [Cantoni Biochem Prep 5 58 1957.] The purity of SAM can be determined by paper chromatography [Cantoni J Biol Chem 204 403 1953 Methods Enzymol 3 601 1957], and electrophoretic methods or enzymic analysis [Cantoni and Vignos J Biol Chem 209 647 1954]. 

Electrophoretic and isotachoelectrophoretic techniques are gaining in popularity in soil analysis with applications to polyaromatic hydrocarbons, polychlorobiphenyls, tetrahydrothiophene and triazine herbicides, Paraquat and Diquat and growth regulators. Other lesser-used techniques include spectrophotometric methods (five determinants), spectrofluorimetric methods (two determinants), luminescence methods (one determinant), titration methods (one determinant), thin-layer chromatography (five applications), NHR spectroscopy (two applications) and enzymic immunoassays (one determinant). 

Acid dye method for the analysis of thiamin, 18A, 73 electrophoretic separation and fluorometric determination of thiamin and its phosphate esters, 18A, 91 catalytic polarography in the study of the reactions of thiamin and thiamin derivatives, 18A, 93 preparation of thiamin derivatives and analogs, 18A, 141 preparation of the mono- and pyrophosphate esters of 2-methyl-4-amino-5-hydroxymethylpyrimidine for thiamin biosynthesis, 18A, 162 formation of the pyrophosphate ester of 2-methyl-4-amino-5-hydroxymethylpyrimidine by enzymes from brewers yeast in thiamin biosynthesis, 18A, 203 resolution, reconstitution, and other methods for the study of binding of thiamin pyrophos-... 

Cycling protein phosphorylation plays a role in the mammalian circadian clock as well. PERI, PER2 and BMAL all show temporal changes in electrophoretic mobility that are eliminated by phosphatase treatment (Lee et al 2001). Although these PER phosphorylations are likely to reflect CKl activity they may not be the only clock-related substrates of this enzyme family. CRYl and CRY2, for instance, can be phosphorylated by CKls in vitro when present in a CRY/PER/CKle complex (Eide et al 2002). Two isoforms of mammalian CLOCK (orthologue of Drosophila CLK) also appear to be phosphorylated, resulting in mobility shifts by Western analysis (Lee et al 2001). The kinase(s) responsible for CLOCK phosphorylation is (are) unknown. 

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