Enzyme analysis

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]. 

Kinetics is the branch of science concerned with the rates of chemical reactions. The study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they accomplish their remarkable feats. In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors. Coupled with studies on the structure and chemistry of the enzyme, analysis of the enzymatic rate under different reaction conditions yields insights regarding the enzyme s mechanism of catalytic action. Such information is essential to an overall understanding of metabolism. 

At present, several of the instruments which are being utilized for enzyme analysis, such as the centrifugal analyzers (15), have been measuring samples of the order of 5 pi. 

McCleary, B.V., and Matheson, N.K. (1986) Enzymic analysis of polysaccharide structure. Adv. Carbohydr. Chem. Biochem. 44, 147-276. 

For those laboratories routinely performing marker enzyme analysis, it is hoped that future investigators will be more attentive to the preparation of balance sheets (24), i.e., comparing the specific activities of marker enzymes in each organellar fraction relative to those in the total cellular homogenate. Unfortunately, some investigators assay for the presence or absence of marker enzymes only in the fraction(s) that he/ she is interested in. 

In most cases the electronic connection between an immobilized redox enzyme and the electrode requires a mediator to shuttle the electrons to the prosthetic group or some type of wiring that plays the same role. There are cases, however, especially those involving relatively small enzymes, where direct electron transfer takes place between the electrode and the prosthetic group or some electronic relay in the enzyme. Analysis of the catalysis responses then follows the principles described and illustrated in Section 4.3.2. Somewhat more complicated schemes are treated in references7, where illustrative experimental examples can also be found. 

Phosphoinositase C (i.e. phosphoinositide-specific phospholipase C [PLC]) enzymes are found in the vast majority of mammalian cells. Molecular cloning of these enzymes, analysis of their predicted amino acid sequences and immunological cross-reactivity indicate that at least three major forms of the enzyme exist PLC-/I, -8 and -y. Each of these enzyme types is encoded by a distinct gene. More recent experiments using the polymerase chain reaction and molecular cloning have revealed even greater enzyme di-... 

Of the many areas where these methods have been useful, the greatest impact has been in the area of complex plant and animal oligosaccharides, glycopeptides, and other glycoconjugates (see Table IV). The isolation of these pure carbohydrates, by the methods described, has allowed their spectroscopic, chemical, and enzymic analysis, in many cases for the first time (see Addendum). 

Methods of Enzymic Analysis, Vol. 3 Enzymes 1 Oxidoreductases, Transferases , Ed. Moss, D. W. Verlag Chemie Weinheim, 1983. 

All of the above-mentioned patterns are specific for the particular disease. The method is suitable as an initial screen to identify those patients in whom such a disorder must be excluded. The diagnosis must then be confirmed by enzyme analysis in serum, leucocytes or cultured skin fibroblasts or by way of mutation analysis. 

Sialic acid determination in cultured fibro- Enzyme analysis, sialidase, /1-galactosidase blasts... 

Mutation screening methods involve testing for specific (previously selected) mutations in a gene. This approach is relatively inexpensive and may be useful for disorders that are caused by one or few common mutations. It is important to take the origin of the patient into consideration, since the frequency of mutations differs markedly between populations. As an example for such a method we discuss restriction enzyme analysis in more detail in this chapter. 

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