Isocitrate dehydrogenase (IDH) exists in mammalian tissues as three enzymes, one specific for NAD and two specific for NADP. The two NADP isozymes differ in electrophoretic mobility, immunological characteristics, and tissue and subcellular location. One is found mainly in the mitochondrial fraction and one in the supernatant fraction of tissue homogenates. Two allelic forms of the supernatant NADP isocitrate dehydrogenase have been described in inbred strains of mice. In a heterozygote containing both alleles, three forms of the supernatant NADP-IDH are generated in a ratio of 1:2:1. This implies that the supernatant enzyme is a dimer. The mitochondrial form is not affected by the gene, indicating separate genetic control. Liver mitochondria contain both the supernatant and the mitochondrial isozymes. Thus at least one protein found in the mitochondria is encoded in a nuclear gene.Research in biochemical genetics led to the formulation of the one-gene-one enzyme hypothesis with the consequent expectation that one enzyme would exist as a single molecular type within an organism. Exceptions would be diploid organisms carrying allelic genes for the enzyme, in which case two forms of the enzyme would be found. However, in recent years an increasing amount of evidence has shown that many enzymes exist in multiple molecular forms, or as isozymes, within the cells of a single organism, even when that organism is homozygous (Markert and Mller, '59; Wieland and Pfleiderer, '57; Vessel1 and Bearn, '58). Such molecular heterogeneity has been detected by many different techniques, including electrophoresis, column chromatography, centrifugation, salt fractionation, and by determination of tissue specific differences in enzyme kinetics, coenzyme specificity, and immunochemical behavior. The moleculuar basis for this heterogeneity may be different for different enzymes. The binding of small molecules to the enzyme can give rise to multiple forms, as in the case of the two active forms of phosphoglucomutase, which differ in the presence of a phosphate group bound to one form during the enzymatic reaction (Koshland, '64 be polymers of different numbers of the same polypeptide subunit. The two forms of phosphorylase, a and b, differ in that one is a dimer of the other as well as in the presence of two phosphate groups bound to serine residues in the dimer (Brown and Cori, '61).On the other hand, the several forms of an enzyme may differ significantly in primary protein structure, and thus be under multiple gene control, as has been clearly shown for the five principal isozymes of lactate dehydrogenase (LDH) . These isozymes are found in characteristic relative amounts in the different tissues of an organism (Markert and MBller, '59) and have the same molecular weight but differ in electrophoretic mobility. The two extreme forms, LDH-1 and LDH-5, differ in amino acid content, immunological specificity, kinetic properties, coenzyme specificity, heat stability, and other characteristics. The intermediate forms, , are also...

No abstract

No abstract

SummaryA tracheal ring explant system, when used with 25% cystic fibrosis (CF) serum, displayed obvious ciliostasis. Hamster, rabbit, and guinea pig explants all had measurable decreases in ciliary activity after 24 hr of incubation in the serum. The differential response to CF serum (relative to normal serum) was greatly increased by using explants which were maintained 24-72 hr in i*dnimal essential medium (MEM) with 10% horse serum and which were selected on the basis of optimal ciliary activity and vigor. With such a bioassay system of guinea pig tracheal explants, incubation with 25% normal serum would produce essentially no change in relative ciliary activity (score of 242 of a possible 300), whereas CF serum resulted in an 86% decrease (score of 33). Scanning electron microscopic observation indicated that the explants displaying the CF-ciliostatic effect had significant accumulations of mucous over the ciliated epithelial surface. A biochemical viability assay (dehydrogenase activity) showed no cytonecrosis when CF serum-treated tissues were compared to standard explants (10% horse serum in MEM) or control explants (25% normal human serum). SpeculationCystic fibrosis serum, when tested at 25% concentrations on preselected guinea pig tracheal ring cultures, showed an obvious and easily measurable ciliostatic response. This new bioassay may serve as an improved model to detect and analyze the CF dyskinesia factor.

Several isozymes of acetylcholinesterase are separated by 10% acrylamide gel electrophoresis of mouse blood, brain, heart, muscle and tongue tissues. Two isozymes migrating near the origin are described which show changes in relative activity during development. The faster of the two bands is proportionately higher in concentration in embryonic tissues and is highly specific for the acetylthiocholine iodide substrate. This isozyme corresponds to the erythrocyte membrane AChE in electrophortic mobility and substrate specificity. The slower of the two bands is predominant in adult tissues and exhibits considerable cross reaction with the butyrylthiocholine iodide substrate. During embryonic and postnatal developmental stages there is a gradual shift from the faster migrating isozyme toward a predominance of the slower migrating isozyme.

Numerous studies with lower eukaryotes such as yeast and Neurospora have shown that mitochondrial morphology and enzyme levels change markedly in response to changes in oxygen tension. Several studies with mammalian cells in culture have indicated that these cells also respond to low oxygen with changes in the levels of certain enzymes. In this study gel electrophoretic analysis of enzymes and electron microscopic analysis of cell organelle morphology were used to study the effects of anaerobic conditions on cultured human embryo fibroblasts. Lactate dehydrogenase showed a marked shift from LDH-1 toward LDH-5 as has been previously described. Isocitrate dehydrogenase, NAD-malate dehydrogenase, and NADP-malate dehydrogenase all have been shown to exist as separate supernatant and mitochondrial isozymes. However, none of these enzymes showed extensive changes in isozyme patterns i n response to anaerobiosis. Electron microscopic examination of cell cultures revealed striking changes in mitochondrial structure. Aerobically grown cells have well-developed, elongated mitochondria with numerous well-defined cristae and a n electron-dense matrix. In cells cultured anaerobically the mitochondria show degenerative changes. They are more rounded, have few and poorly defined cristae, and the matrix is more electronlucent. These studies indicate that, as in lower eukaryotes, the mitochondria of cultured human cells also respond morphologically and biochemically to changes in oxygen tension.

Isozyme patterns of cholinesterase (ChE) from heart, tongue, and skeletal muscle of normal and dystrophic hamsters are presented. Two principal bands, bands 1 and 2, were evaluated. Band 1 migrates faster towards the anode than does band 2. While bands 1 and 2 stain for AChE and were found in control muscles, only band 2 was stained by a pseudocholinesterase (BuChE) and was decreased in samples from dystrophic hamsters. The decrease in BuChE was most pronounced in dystrophic heart muscle. The low level of BuChE measured for dystrophic animal tissue was similar to isozyme patterns found in embryonic tissue and in denervated muscle. BuChE obtained by acrylamide gel electrophoresis along with 16S AchE appears to be a useful biochemical marker of nerve-muscle interactions.