Phospholipase solutions

Phosphatidylcholine Apply phospholipase C solution as a band, dry, apply sample solution to enzyme band, stop reaction with hydrochloric acid vapor. sn-l,2-Digly-cerides are produced. [43]... 

Barium, effectiveness as cofactor for, see also Enzyme cofactors phospholipase, 204 SNase, 200-204 Bond-breaking processes, 12 potential surfaces for, 13-14, 18-20 in solutions, 22,46-54... 

A common problem with gap junction measurements is a rundown of gj in these preparations, for example in neonatal rat heart cells Schmilinsky-Fluri et al. [1990] found a decrease in g, of 16.4% in 6 min which could be antagonized by addition of a phospholipase inhibitor, 20 pmol/l bromophenacyl bromide, to 1.8% within 6 min. They suggested that endogenous arachidonic acid is involved in spontaneous uncoupling. Others favored a washout of ATP and cyclic nucleotides as a possible cause and prevented their preparations from spontaneous uncoupling by addition of ATP, GTP or cAMP to the pipette solution [Miiller et al., 1997a, b]. 

In a typical reaction, 10-15 mg of phosphatidylcholine (egg lecithin is a typical substrate) is dissolved in 2-2.5 ml of diethyl ether-methanol (95 5, v/v) and transferred to a 5-ml glass-stoppered volumetric flask. To this clear solution is added 20 jjlI of an aqueous solution containing 20 mM Ca2+ and approximately 3 xg of purified phospholipase A2. The flask is stoppered and the contents are mixed well for 4-5 min and then allowed to stand at room temperature for 30-45 min. Usually a precipitate forms during the course of the reaction but as noted before, this does not compromise the extent of the reaction. 

As mentioned in an earlier section, there are no chemical reagents that can establish with any certainty the specific position of attachment of an acyl ester on a diacylphosphatidylcholine or any other phosphoglyceride. The same is true in any attempt to establish the stereochemical conformation of phosphatidylcholine, whether sn-1 or sn-3, by strictly chemical means. The solution to this dilemma is to use synthetic phosphoglycerides of defined structure as well as phospholipase A2 to establish the correct stereospecificity of the latter enzyme. Certainly, this is reminiscent of the chicken or egg argument, yet it does work as will be explained below. 

The water-methanol-rich lower phase is treated with an alkaline solution to convert the sphingosine acid salt to a neutral form. This mixture is extracted with diethyl ether to recover the free sphingosine, which can then be analyzed further. The water-rich phase from the latter extraction can be analyzed for choline as described in Chapter 3. The choline/P molar ratio should be 1.0. An alternative route to the release of 0-phosphocholine and choline by action of phospholipases C and D, respectively, will be considered below. 

An important observation was made in the de Haas laboratory in Utrecht, where the activities of the pancreatic phospholipase and its proenzyme on the same substrate have been tested (22). The phospholipase hydrolyzes substrates that are part of a micelle, but the prophospholipase is completely inactive against micelles. The phospholipase can also hydrolyze short chain phospholipids that are in monomolecular solution although at much slower rates. The prophospholipase is almost as active as the enzyme against such substrates. It must be concluded that the reactive site of the phospholipase is already completely assembled and active in the proenzyme. Tryptic activation forms a new site that binds to micelles. This site is called the anchoring site or recognition site (22). 

With this structure in mind, we can now understand how PKC is activated on PIP2 hydrolysis (Figure 15.16B). Before activation, PKC is free in solution. On PIP2 hydrolysis in the membrane by phospholipase C, the CIB domain of PKC... 

Despite their different physical and biochemical properties, most lipases and phospholipases share a common structural element an a-helical loop ( lid ) that covers the active site. Since the opening of the lid exposes a large hydrophobic patch, the resulting open conformation is thermodynamically unfavorable in solution. In contrast, in the presence of a lipid interface the open conformation is stabilized by the interaction with lipids. Many lipases and phospholipases show higher activity on interfaces than with free lipids (interfacial activation). It has long been considered that interfacial activation and lid opening are correlated. However, a number of enz3unes, such as CalB, possess a lid structure but do not show interfacial activation [18-20]. 

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