Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.
Use of antipeptide antibodies plus a panel of monoclonal antibodies to screen the testicular lactate dehydrogenases should provide highly sensitive discrimination of their evolutionary relatedness. When this is combined with sequence information and peptide synthesis, it will be possible to describe the precise limits of each epitope on the surface of LDH-C4. From the standpoint of contraceptive vaccine development, a synthetic antigen consisting of one or more epitopes can be prepared. This will eliminate the necessity of attempting to isolate antigens from natural products, and will provide a useful strategy for developing this approach to fertility control.
The principal means by which genetic variation of enzyme proteins has been revealed is the physicochemical separation of isozymes in an electrical field followed by their specific detection. This approach is widely applicable to all types of species and almost every conceivable kind of tissue homogenate or protein secretion. In the human organism alone the products of more than 200 enzyme loci have been investigated by this means and more than 40 genetically determined human isozyme polymorphisms have been identified. Much of this progress has been achieved by the use of conventional gel electrophoresis methods and isozyme detection schemes that depend on enzyme-catalyzed conversion of substrate into chromogens or other types of visible product. However, the functional isozyme detection systems are often of limited sensitivity and are dependent on the good condition of the biological material under analysis and the retention of adequate enzyme activity. Also in some cases, especially where the final product of the detection reaction is not an insoluble compound, the staining may be so diffuse as to offset the high resolving power of the most effective protein separation methods such as isoelectric focusing in ultranarrow pH gradients. The recent development of alternative protein detection methods based on immunoblotting offers a new general approach to isozyme analysis and overcomes some of the limitations of isozyme detection by functional staining methods. The immunoblot methodology is applicable to proteins separated electrophoretically on denaturing gels and on native gels, including proteins separated by isoelectric focusing, and the fidelity of the high-resolution separation techniques appears to be preserved for most proteins by passive or electroblot transfer to nitro-cellulose filters. Furthermore this approach provides considerable flexibility in the sensitivity and the nature of the final reporter signal that is employed. For example, the reporter signal can be amplified by the use of appropriate intermediate reagents to provide a level of detection sensitivity for isozyme proteins which should, even with the present procedures, be far in excess of that which is obtained even by the best functional isozyme staining system. Also the use of an evanescent reporter system such as chemiluminescence may allow multiple analyses of the same immunoblot and hence examination of several different isozyme systems by sequentially using different primary antibodies.
The metabolism of acetaldehyde has received considerable attention in the past owing to its acute and chronic toxic effects in humans. Two major hepatic ALDH isozymes, ALDH I and ALDH II, differing in their structural and functional properties, have been characterized in humans. ALDH I has a low Km for acetaldehyde and is primarily a mitochondrial enzyme, while ALDH II has a higher Km and is of cytosolic origin. An inherited deficiency of ALDH I isozyme found only among Oriental populations is primarily responsible for producing acute alcohol sensitivity symptoms (flushing response) after consumption of small doses of alcohol. Biochemical, immunochemical, and molecular genetics data indicate a structural mutation in the ALDH I isozyme gene responsible for the loss in catalytic activity. Population genetic studies have revealed the prevalence of ALDH polymorphism among individuals of the Mongoloid race. Flushing response to alcohol is a familial trait, and preliminary family data from Japan, China, and Korea suggest an autosomal codominant inheritance for ALDH I isozyme deficiency. The ALDH polymorphism is apparently responsible for the low incidence of alcoholism in Japanese, Chinese, and Koreans. Alcohol sensitivity due to ALDH I isozyme deficiency may inhibit excessive alcohol drinking.
Linkage or association of genetic markers to quantitative traits of agronomic importance can substantially simplify the genetic analysis of complex quantitative traits. Enzyme marker genes are ideal candidates for quantitative genetic analysis. We have recently applied this powerful methodology to the analysis of genetic resources of wild cereals in Israel, primarily wild emmer wheat, Triticum dicoccoides, and wild barley, Hordeum spontaneum, the progenitors of cultivated wheats and barley, respectively. We have found that allelic isozyme markers and ecological factors provide an important predictive method for identifying elite genotypes characterized by single or multiple disease resistances, high protein content, and a variety of quantitative traits of agronomic importance including germination, earliness, biomass, and yield variables. Our predictive methodology could be improved by additional crossing tests in an attempt to verify the results derived from the correlation analysis and thus establish the linkage relationships between the marker gene and the quantitative trait under discussion. This methodology, if further developed by additional isozyme and DNA markers, verified by crossing tests and gene mapping, could substantially contribute to the sampling and utilization of the genetic resources of wild gene pools for crop improvement.
Les nucléosides et nucléotides extracellulaires sont maintenant reconnus comme des molécules de signalisation au même titre que les neurotransmetteurs et les hormones. Les cellules hépatiques, comme la majorité des autres cellules de l'organisme, expriment plusieurs récepteurs transmembranaires spécifiques ainsi que plusieurs ectoenzymes dont ces médiateurs sont les ligands et les substrats, respectivement. Cette revue de littérature présente l'état des connaissances actuelles sur la signalisation par les nucléosides et nucléotides extracellulaires dans le foie (en y incluant les sources, la transduction de signal et le métabolisme), ainsi que sur leur contribution dans le maintien de l'homéostasie cellulaire hépatique.Nucleosides and nucleotides are now considered as extracellular signalling molecules, like neurotransmitters and hormones. Hepatic cells, amongst other cells, ubiquitously express specific transmembrane receptors that transduce the physiological signals induced by extracellular nucleosides and nucleotides, as well as various cell surface enzymes that regulate the levels of these mediators in the extracellular medium. Here, we cover various aspects of the signalling pathways initiated by extracellular nucleosides and nucleotides in the liver, and discuss their overall impact on hepatic physiology.
According to the nomenclature of Vallee and Bazzone [1983] for mammalian alcohol dehydrogenase (ADH) isozymes, baboon ADHs comprise three major classes of activity, which were distinguished according to the following properties: Class I ADHs. These isozymes exhibited low-Km characteristics with ethanol as substrate, high isoelectric points (8.5-9.3), and sensitivity to 5 mM 4-methyl pyrazole inhibition, and were the major liver (ADH-2) and kidney (ADH-1) isozymes in the baboon. Class II ADHs. These isozymes showed high-Km values for ethanol, neutral isoelectric points (7.7 for the liver ADH-4 [pi-ADH] and 7.2 for the major stomach ADH [ADH-3], respectively), and were insensitive to inhibition with 5 mM 4-methyl pyrazole. Class III ADH. This enzyme was characterized by its inactivity with ethanol as substrate (up to 0.5 M), insensitivity to 4-methyl pyrazole inhibition, preference for medium-chain-length alcohols as substrate (trans-2-hexen-1-ol was routinely used in this study), and an isoelectric point (6.5) similar to that of the human liver chi-ADH (pI 6.4). Major activity variation of the liver pi-ADH (ADH-4) isozyme was observed among the 114 liver samples examined, with 34 percent exhibiting a null (or low-activity) phenotype. An electrophoretic variant phenotype for the major class II stomach isozyme (ADH-3) was also found in the population studied. The baboon was used as a model for studying alcohol-induced changes in liver ADH phenotype following chronic alcohol consumption. Prepuberal male baboons were pair-fed nutritionally adequate liquid diets containing ethanol (50 percent of calories) or isocaloric carbohydrates, and liver ADH isozyme patterns from biopsy samples were monitored for 20 weeks. Dramatic decreases in class II liver ADH activity (ADH-4, or pi-ADH) were observed within 4 weeks after the start of alcohol feeding, and a shift in liver class I isozymes was found during the later stages of alcohol consumption. These changes during chronic alcohol consumption may be adaptations of the liver: these include reduced capacity of the major ADH pathway and increased ethanol oxidation by the microsomal ethanol oxidizing system and possibly by peroxisomal catalase.
