Enzymes marker

The deterrnination of the presence of reverse transcriptase in vims-infected cells can be done using labeled nucleotide triphosphates. Reverse transcriptase is an enzyme capable of synthesizing DNA from RNA and it is thought to play an important role in vims-mediated cell modification. This enzyme is also a marker enzyme for HIV, the vims impHcated in causing acquired immunodeficiency syndrome (AIDS). The procedure utilizes radiolabeled nucleotides with nonlabeled substrates to synthesize tagged DNA. The degree of radioactive incorporation reflects the reverse transcriptase activity. 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

Hours after inoculation of self-induced anaerobic culture. Organelle identified by peak of marker enzyme activity. 

The effect of the material on cellular metabolism is also an important measure of biocompatibility. To determine such effects, cultured ex vivo cells can be exposed to the polymer and the growth rates compared to controls [216,217], The metabolic function of the cells can be tested by assay for production of a marker enzyme. An additional advantage of this type of test is that it avoids the use of live animals. 

Porstmann, B., Porstmann, T., Nugel, E., and Evers, U. (1985) Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays Horseradish peroxidase, alkaline phosphatase or b-galactosidase. J. Immunol. Meth. 79, 27-37. 

In the method shown in Figure 9A, a biotin-labeled cDNA probe is first immobilized to a polyvinylchloride microtiter plate well that is coated with bio-tinylated-bovine serum albumin [33], The target DNA is hybridized in the liquid-phase with a digoxigenin-labeled probe, so that the biotin-labeled probe can capture a marker enzyme. An antibody-conjugated enzyme is then added, followed by a chemiluminescent substrate. 

Fung and colleagues examined the metabolic conversion of organic nitrates in sub-cellular fractions of bovine coronary artery smooth muscle cells [66, 67]. They found NO-generating capacity to be present in membrane fractions and, with the use of marker enzymes, identified plasma membrane as the primary location. The enzyme involved in bioconversion was not glutathione-S-transferase [68] and differed from those that catalyse activation of organic nitrites [69]. Partial purification [70] established that the molecular sizes of the native enzyme and subunits were approximately 200 kDa and 58 kDa respectively, and that enzymic activity depends on the presence of a free thiol group. 

Most of the intact mitochondria and chloroplasts may be removed at the first centrifugation. However, a large amount of these organelles still existed in the microsomal fraction as estimated from the results of marker enzyme assays. 

It is well-established that marker enzyme activity is an index of organelle (or organelle-related component) existence. In this experiment, assays of marker enzymes, which are localized in organelle membranes, are carried out as described below. 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

The microsome fractions see Fig. 1) that were prepared from mulberry cortical parenchyma cells were fractionated to 24 or 25 fractions using the 15-50% sucrose linear density gradient centrifugation see Fig. 2). Profiles of the marker enzymes and the protein content are described in Fig. 3. In general, the antimycin A-insensitive cytochrome c reductase activity is exhibited at a lower density than are those of the marker enzymes. The fraction that exhibited the highest antimycin A-insensitive cytochrome c reductase activity for each month was used as the ER-enriched fraction. 

Fig. 4. Localization of WAP27 and WAP20 in the crude microsome fractions and the relation with marker-enzyme activities in three organelles (ER, tonoplast, and Golgi). SDS-PAGE of fractionated proteins by isopycnic linear sucrose density gradient centrifugation of microsome fraction of mulberry cortical parenchyma cells was performed using 6-pL samples in each fraction. Immunoblot analysis was performed with anti-WAP27 and anti-WAP20 antibodies. (From ref. [1], with permission from the American Society of Plant Physiologists.)...

Assay of Inosine-5-diphosphatase (IDPase) EC 3.6.1.6, Presumed Marker Enzyme for Golgi Apparatus Conclusions References... 

Marker Enzymes Table 1 Useful in the Identification of Plant Organelles ... 

Membrane Equilibrium density in sucrose g/cm3 %w/w Marker enzyme/substance... 

Adapted from Robinson et al. (5). Examples of other marker enzymes can be found in Bowles et al. (1). 

Table 2 References for Assay Conditions of Significant Marker Enzymes ... 

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. 

Halliwell B. Marker enzymes of plant cell organelles, in Methodological Developments in Biochemistry. 4. Subcellular Studies (Reid E, ed.), Longman, London, pp. 357-366. 

Two studies were located in which rats received di- -octylphthalate dietary exposures of 250 or 500 mg/kg/day for either 10 or 26 weeks (Carter et al. 1992 DeAngelo et al. 1986). Five male rats were first initiated with a single subcarcinogenic intraperitoneal dose of diethylnitrosamine (30 mg/kg), followed by partial hepatectomy. Di-w-octylphthalatc caused substantial increases in gamma-glutamyltranspeptidase (GGT) positive liver foci when compared with the controls (e.g., from 3.5 to 20.8 foci/cm2) or in hepatic levels of marker enzymes for altered cellular foci (GGT and glutathione 5-transferase [GST]). Only a slight increase (threefold) was observed for carnitine acetyltransferase (CAT) activity, a marker for peroxisome... 

Marker enzymes can be used to assess the presence of specific organelles in sub-cellular preparations. 

It is often necessary to assess the efficiency of cell fractionation procedures. Electron microscopy of the prepared fractions is very informative but gives no quantitative indication of the purity of the fraction. It is often easier to measure the relative concentrations of marker enzymes in each fraction (Table 8.9). 

---