Specific activity, of enzymes

The high specific activity of enzymes and tfie tfieoretical possibility of using them to conduct electrochemical reactions are topics of great scientific interest. However, it is difficult to envisage prospects for a practical nse of enzymes for an acceleration and intensification of industrial electrode processes. The difficulty resides in the fact that enzymes are rather large molecnles, and on the surface of an enzyme electrode, fewer active sites are available than on other electrodes. Per unit snrface area, therefore, the effect expected from the nse of enzymes is somewhat rednced. 

Biochemical studies have also suggested an asymmetric orientation of constituents in lipid globule membrane. By comparison of specific activities of enzymes in washed lipid globules and released membrane, Patton and Trams (1971) suggested that the active site of Mg2+-adeno-sine triphosphatase was accessible to substrates on both faces of the membrane and that of 5 -nucleotidase on the outer membrane face. Recent evidence from studies of Concanavalin A inhibition of globule membrane and plasma membrane 5 -nucleotidase support an outer surface localization for the active site of this enzyme (Carraway and Carra-way 1976 Snow et al. 1980). Kobylka and Carraway (1973) observed that exposure of lipid globules to proteolytic enzymes resulted in cleavage of all major membrane-associated proteins. They concluded that... 

Parenthetically, it should be noted that mitochondrial respiration rates normalized to mitochondrial protein content do not exhibit a significant degree of compensation to temperature (see Johnston et al., 1994). For mitochondria, then, changes in membrane physical state linked to homeophasic and homeoviscous adaptation do not appear linked to temperature-compensatory changes in specific activities of enzymes. 

D-Glucose D-Galactose D-Glucose D-Galactose Specific activities of enzymes (in nmoles of substrate Enzyme reacted/minute/mg of protein)... 

The specific activity of enzymes is given in units. One international unit (IU) is the amount of enzyme consuming or forming 1 pmol substrate or 1 pmol product per minute under standard conditions. The base unit is 1 katal, corresponding to the amount of enzyme converting 1 mol substrate per second ... 

Table 1 Specific activities of enzymes invoived in the biosynthesis of riboflavin of Escherichia coli...

The finding that the specific activities of enzymes in the mitochondrial fraction from whole embryos do not change during development is rather surprising. It implies that (1) the relative contributions of mitochondria, with different specific activities, remain constant during development ... 

Fig. 5. (A) Specific activities of enzymes of mitochondria from rat brown adi-...

Some typical results on changes in the specific activities of enzymes from mitochondria in which the amount of inner membrane increases during development are shown in Figs. 7 and 8. In these mitochondria, as Table IV shows, the amount of inner membrane, reckoned as insoluble protein, increases approximately 2-foId. The specific activities of cytochrome oxidase and succinate dehydrogenase also increase by about the same relative amount. These data suggest that the enzymic activity per unit of membrane is constant during development, and the calculations summarized in Table V show that this is the case. 

Usually, the protein content of tissues or samples is not a major research interest but serves as a reference quantity. As a consequence it is desirable to perform protein determination with the least effort. Nevertheless, care should be taken to obtain correct results, especially when protein-related data (e.g., specific activities of enzyme preparations, yields in protein purification) are calculated. This article focuses on three techniques and outlines the specific pros and cons. With respect to convenience and speed, microplate reader assays are described where appropriate. These assays can be easily read in conventional instruments by employing microcuvettes or by scaling up the volumes (fivefold). 

The higher activity in frosthardened chloroplasts could be due to an augmentation of envelope membranes or to increased specific activity of enzymes. The galactolipid biosynthesis of frosthardened chloroplasts at 7C was only slightly lower than that of unhardened chloroplasts at 20C. 

For purposes of dosage, the specific activity of an enzyme is usually expressed as International Units (lU) rather than in terms of weight. However, unit measurements do not provide information on the absolute purity of a given product. Moreover, purity is not as critical an attribute for oral enzymes, as opposed to those adrninistered parenteraHy, inasmuch as the gastrointestinal tract is capable of disposing of most inert contaminants. 

The biochemical basis of penicillin action continues to be an area of active investigation. Penicillins are highly specific inhibitors of enzyme(s) involved in the synthesis of the bacterial cell wall, a structure not present in mammalian cells. Three principal factors are thought to be important for effective antibacterial action by a penicillin ... 

Most purification procedures for a particular protein are developed in an empirical manner, the overriding principle being purification of the protein to a homogeneous state with acceptable yield. Table 5.5 presents a summary of a purification scheme for a selected protein. Note that the specific activity of the protein (the enzyme xanthine dehydrogenase) in the immuno-affinity purified fraction (fraction 5) has been increased 152/0.108, or 1407 times the specific activity in the crude extract (fraction 1). Thus, xanthine dehydrogenase in fraction 5 versus fraction 1 is enriched more than 1400-fold by the purification procedure. 

Ca2+-pumps. After entering the cell, Ca2+ is reversibly complexed to specific Ca2+-binding proteins that fiilfil multiple functions, including Ca2+-buffering and transport, activation of enzymes, regulation of contraction,... 

The class I FruA isolated from rabbit muscle aldolase (RAMA) is the aldolase employed for preparative synthesis in the widest sense, owing to its commercial availability and useful specific activity of 20 U mg . Its operative stability in solution is limiting, but the more robust homologous enzyme from Staphylococcus carnosus has been cloned for overexpression [87], which offers unusual stability for synthetic purposes. Recently, it was shown that less polar substrates may be converted as highly concentrated water-in-oil emulsions [88]. 

The minute quantities of enzymes present in cells complicate determination of their presence and concentration. However, the abifity to rapidly transform thousands of molecules of a specific substrate into products imbues each enzyme with the abifity to reveal its presence. Assays of the catalytic activity of enzymes are fre-quendy used in research and cfinical laboratories. Under appropriate conditions (see Chapter 8), the rate of the catalytic reaction being monitored is proportionate to the amount of enzyme present, which allows its concentration to be inferred. 

Inhibitor assay A suitable amount of inhibitor was preincubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for 10 minutes at 37°C. Control without inhibitor was run simultaneously. The enzyme reaction was initiated by the addition of 1 ml of substrate solution (1% polygalacturonic acid). The decrease in PG activity was a measure of the inhibitory activity. Proper controls containing only Dieffenbachia extract and no fungal PG in the assay mixture were also run to account for the inherent PG activity, if any, of Dieffenbachia extract. One unit of inhibitor activity is defined as the amount of inhibitor that reduces the polygalacturonase activity under the assay conditions by one unit. Specific activity of the inhibitor is expressed as units per mg protein. 

---