In this protocol, we describe an approach in which two-dimensional electrophoresis (2DE)-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminally sulfonated without any further sample purification. The protocol was applied on a proteomic study of 2DE-separated p...

A procedure has been developed whereby the components of the nitrate reductase complex from the membrane of Escherichia coli can be extracted by means of a nonionic detergent and separated from each other as discrete elements in an active form. It is not yet clear whether these are the dissociated enzymatic elements or whether they are fragments containing other membrane proteins. In any case, ...

The cell wall lipopolysaccharide of Escherichia coli 011 l-B4 was degraded by (a) acid hydrolysis and (b) by treatment with periodate, followed by reduction with sodium borohydride and mild acid hydrolysis. The various oligosaccharides produced by these procedures were isolated and characterized by chemical and enzymatic methods. Procedure (I released all of the colitose (3,6-dideoxy-~-xylohexo...

We describe a technique whereby intracellular urease activity can be localized by transmission electron microscopy. The ammonia produced from the enzymatic hydrolysis of urea is first precipitated with sodium tetraphenylboron and then replaced with silver to produce electron-dense silver tetraphenylboron. This direct reaction product deposition procedure was used to demonstrate the presence of ...

The cell wall lipopolysaccharide of Escherichia coli 011 l-B4 was degraded by (a) acid hydrolysis and (b) by treatment with periodate, followed by reduction with sodium borohydride and mild acid hydrolysis. The various oligosaccharides produced by these procedures were isolated and characterized by chemical and enzymatic methods. Procedure (I released all of the colitose (3,6-dideoxy-~-xylohexo...

Cyclohexane analogues of the antifungal icofungipen [(1R,2S)-2-amino-4-methylenecyclopentanecarboxylic acid] were selectively synthesized from unsaturated bicyclic β-lactams by transformation of the ring olefinic bond through three different regio- and stereocontrolled hydroxylation techniques, followed by hydroxy group oxidation and oxo-methylene interconversion with a phosphorane. Starting fr...

The nitrate reductase complex from Chlorella pyrenoidosa has been purified by a procedure which includes as main steps, ammonium sulfate fractionation, polyethylene glycol treatment, and DEAE-cellulose chromatography. The Michaelis constants for NADH, FAD, and NO(3) (-) in the NADH-nitrate reductase assay are 10 mum, 2.6 mum, and 0.23 mm, respectively. Heat treatment exerts varying effects on t...

A method for the determination of serum arginase is given which combines the enzymatic formation of urea with the sensitive method of Coulombe (1) for measuring this substance. This procedure allows more accurate determinations in the normal range than do previous methods described and is convenient for clinical routine. Significant difference is found between the mean values of normal men and ...

These enzymatic reactions have been utilized for t,he preparation of RuDP. In the present paper the isolation procedure and proof of structure of RuDP are described, together with some of the properties of this compound. The availability of substrate quantities of RuDP has permitted a direct study of its reaction with CO2 and the purification of the carboxylation enzyme.2 This work will be repo...

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenot...