Molecular properties isoenzymes

Tannenbaum, S. W., and Sun, S.-C., 1971, Some molecular properties of pneumococcal neuraminidase isoenzymes, Biochim. Biophys. Acta 229 824-828. 

The psubunit has been purified from PGl by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The psubunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PGl. Purified psubunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PGl (13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PGl by the association of PG2 with the p-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PGl and PG2 in total cell wall protein extracts. 

In M. rhodesianum no isoenzymes of the 3-ketothiolase have been found [ 14]. The enzyme s capability to react with long-chain acyl-CoAs has not been tested. The enzyme was found to be very similar to the 3-ketothiolases from R. eutropha, A. beijerinckii, Z. ramigera, and R. ruber with respect to molecular weight, optimum pH, and kinetic properties [14]. 

Communi, D. Vanweyenberg, V. Erneux, C. Purification and biochemical properties of a high-molecular-mass inositol 1,4,5-trisphosphate 3-kinase isoenzyme in human platelets. Biochem. J., 298, 669-673 (1994)... 

The four forms of hexokinase found in mammalian tissues are but one example of a common biological situation the same reaction catalyzed by two or more different molecular forms of an enzyme. These multiple forms, called isozymes or isoenzymes, may occur in the same species, in the same tissue, or even in the same cell. The different forms of the enzyme generally differ in kinetic or regulatory properties, in the cofactor they use (NADH or NADPH for dehydrogenase isozymes, for example), or in their subcellular distribution (soluble or membrane-bound). Isozymes may have similar, but not identical, amino acid sequences, and in many cases they clearly share a common evolutionary origin. 

Hexokinase has a molecular weight of 102,000 and is composed of two identical subunits of 51,000 molecular weight each (20-22). In yeast, hexokinase exists as a mixture of two isoenzymes (23). These forms have been named A and B (23). These isoenzymes can be separated by chromatography and have been found to be chemically different (23, 24). Some of the earlier work with hexokinase was done with preparations that contained a mixture of isoenzymes. Work also was done with enzyme that was proteolytically modified. These variations undoubtedly have been responsible for some of the controversy concerning the properties of this enzyme. 

The second messenger molecules Ca2+ and cyclic AMP (cAMP) provide major routes for controlling cellular functions. In many instances, calcium (Ca2+) achieves its intracellular effects by binding to the receptor protein calmodulin. Calmodulin has the ability to associate with and modulate different proteins in a Ca2+-dependent and reversible manner. Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE, EC 3.1.4.17) is one of the key enzymes involved in the complex interactions that occur between the cyclic-nucleotide and Ca2+ second messenger systems (see Figure 13.2). CaMPDE exists in different isozymic forms, which exhibit distinct molecular and catalytic properties. The differential expression and regulation of individual phosphodiesterase (PDE) isoenzymes in different tissues relates to their function in the body. 

Yoshida, T., Inoue, T., and Ichishima, E. (1993). 1,2-a-D-mannosidase from Penicillium citrinum molecular and enzymatic properties of two isoenzymes. Biochem. J., 290, 349-354. 

The second main class of blood constituents used as genetic markers are the polymorphic enzymes. The enzymes of interest to the forensic serologist are primarily located within the red blood cell and are commonly referred to as isoenzymes. These can briefly be described as those enzymatically active proteins which catalyze the same biochemical reactions and occur in the same species but differ in certain of their physicochemical properties. (This description does not exclude the tissue isoenzymes that occur within the same organism however, our consideration deals only with those of the red blood cell in particular.) The occurrence of multi-molecular forms of the same enzyme (isoenzymes) has been known for several decades however, it was not until the Metropolitan Police Laboratory of Scotland Yard adapted electrophoretic techniques to dried blood analysis that these systems were catapulted to the prominence they presently receive (.2). For many of the forensic serologists in the United States, the use of electrophoresis and isoenzyme determination is a recently-inherited capability shared by only a few laboratories. 

It is now recognised that a substantial number of proteins, especially enzymes, are polymorphic in that they exist in the cell as multiple molecular forms differing in certain of their physico-chemical properties. Each form of a polymorphic enzyme is called an isoenzyme or isozyme. Electrophoretic techniques provide convenient methods whereby this protein heterogeneity can be investigated and the approach has been widely exploited to characterise parasites. In short, aqueous parasite extracts are electro-phoresed, or focused isoelectrically, and separated proteins are stained generally (usually with Coomassie Blue) or more specifically with a histochemical (enzyme) stain (the zymogram technique). Further details of individual procedures and the use of the approach in parasite identification are to be found in a number of recent reviews (104,258,413,536,615,856). 

The purification of acid phosphatase from the human liver and the description of its properties do not appear to have been accomplished. Partly, this may be due to the inherent difiBculty of obtaining normal, fresh human material in amounts substantial enough for purification. However, because of the cellular and physiological importance of acid phosphatase, it is advisable to describe in the present section the purifications of the enzyme from rat and bovine liver. Moreover, since these purifications were accomplished with the awareness that acid phosphatase from this source might be present in multiple molecular forms, the descriptions will naturally involve a consideration of the isoenzymes and their properties. 

The properties of these two components or isoenzymes of rat liver phosphatase were similar in many respects, but different in some. Thus, the molecular weight of each was approximately 100,000. With p-nitro-phenyl phosphate as substrate, the Michaelis constant was 0.091 0.007 mM for the crystalline isoenzyme and 0.047 0.004 for the PII component. The isoelectric points of the crystalline and PII isoenzymes were pH 7.7 and 4.5, respectively, as determined by the method of isoelectric focusing. The activity of each isoenzyme was completely inhibited by 1 mM l-(-f)-tartrate or fluoride at a concentration of 1.0 mM" p-nitrophenyl phosphate as substrate. 

The mammalian hepatic, renal, and intestinal CarbEs consist of units with a molecular weight of 60 000. Each unit bears one active site. The amino acid sequence around the active site of several CarbEs is Gly-Glu-Ser -Ala-Gly. The pi of hepatic CarbEs is usually in the range of pH 4.7-6.5 with the pH optimum in the range of pH 6-10. The behavior of hepatic microsomal and cytosolic CarbEs in in vitro and in vivo studies indicates that these enzymes are different. CarbE in hepatic microsomes consists of three isoenzymes (RHl, molecular weight 174 000, trimer, p7 6.0 RLl, molecular weight 61 000, monomer, p7 6.5 and RLl, molecular weight 61000, monomer, p7 5.5), which differ considerably in terms of inducibility, substrate specificity, and immunological properties. 

Isoenzymes caused by the existence of multiple-gene loci usually differ quantitatively in catalytic properties. These differences may be manifested in such characteristics as molecular activity, K, values for substrate(s), sensitivity to various inhibitors, and relative rates of activity with substrate analogues (when the specificity of the isoenzymes allows the substrate to be varied), underscoring the biological importance of isoenzymatic variation. In contrast, multiple enzyme forms that arise by such posttranslational modifications as aggregation usually have similar catalytic properties. 

The sensitivity to Ca2+ and diacylglycerol and specific binding of phorbol esters has been considered for a long time to be the main characteristics of protein kinase C enzymes. Molecular cloning techniques and biochemical work, however, revealed that protein kinase C is a large kinase family that includes a variety of isoenzymes with very different regulatory properties (see below). 

Feussner, I and Kindi, H. (1994) Particulate and soluble lipoxygenase isoenzymes - comparison of molecular and enzymatic properties, Planta 194, 22-28. 

CAs are ubiquitous metalloenzymes that catalyze the reversible synthesis of bicarbonate ion and proton from carbon dioxide and water. This family includes 15 isoenzymes (1-XlV, V further subclassified as VA and VB) endowed with different molecular features, cellular localization, distribution in organs and tissues, and kinetic properties. It has been demonstrated that the dysfunction of these enzymes is related to different pathologies such as cancer (CAs Vll, IX, XII), neurodegeneration (CA VIII), obesity (CAs VA and VB), and sterility (CA XIII) [36]. Based on the knowledge that coumarin derivatives are CA inhibitors [37], a library of suflbcoumarin derivatives 21 bearing a triazole moiety was synthesized (Scheme 2.3). These compounds 21 (R = Ph) were found to... 

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