Intact spinach chloroplasts scavenge hydrogen peroxide with a peroxidase that uses a photoreductant as the electron donor, but the activity of ruptured chloroplasts is very low [Nakano and Asada (1980) Plant & Cell Physiol. 21: 1295]. Ruptured spinach chloroplasts recovered their ability to photoreduce hydrogen peroxide with the concomitant evolution of oxygen after the addition of glutathione and dehydroascorbate (DHA). In ruptured chloroplasts, DHA was photoreduced to ascorbate and oxygen was evolved in the process in the presence of glutathione. DHA reductase (EC 1.8.5.1) and a peroxidase whose electron donor is specific to L-ascorbate are localized in chloroplast stroma. These observations confirm that the electron donor for the scavenging of hydrogen peroxide in chloroplasts is L-ascorbate and that the L-ascorbate is regenerated from DHA by the system: photosystem I-*ferredoxin-*NADP^>glutathione. A preliminary characterization of the chloroplast peroxidase is given.

Hydrogen Peroxide is Scavenged by Ascorbate-specific Peroxidase in Spinach Chloroplasts

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Evolution at two levels in humans and chimpanzees

Methodes d'extraction d'enzymes, d'electrophorese en gel et de coloration specifique des enzymes utilisees pour etudier la variation genetique chez Escherichia coli et d'autres bacteries. Les procedures decrites peuvent s'appliquer, avec quelques modifications mineures, a toute espece de bacterie

Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics

The homosporous pteridophytes have been largely uninvestigated by electrophoresis, despite the fact that they offer many exciting research possibilities (Soltis et al., 1980). The paucity of electrophoretic studies of ferns and fern allies may be due in large part to the high concentrations of condensed tannins that many species contain (Cooper-Driver, 1976 and pers. comm.). These compounds render enzymes inactive by binding with them following cellular disruption, thereby frustrating researchers who have attempted electrophoretic analysis utilizing standard methods of sample preparation. The method of sample preparation developed by Kelley and Adams (1977a, b) in their analysis of enzyme variation in Juniperus was an important procedural breakthrough in overcoming the difficulties that result from the liberation of large amounts of phenolic compounds during tissue preparation. Recently, a simplified version of that method was applied by Soltis et al. (1980) to fern leaf tissue, facilitating rapid preparation of active enzyme samples and thereby making electrophoretic analyses of large numbers of individuals more feasible. In an attempt to improve methods of analysis of fern enzymes in starch gel electrophoresis, we have experimented with modifications of the method of sample preparation outlined by Soltis et al. (1980). We also have examined several different methods of sample preparation such as those of Gottlieb (1981a), Mitton et al. (1979), and Werth et al. (1982), and have evaluated the relative merits of each with fern tissue. Finally, during the course of our electrophoretic investigations of ferns we found that standard gel and electrode buffers and staining schedules, such as those of Brewer (1970) and Shaw and Prasad (1970), often provided unsatisfactory results when applied to ferns. We have determined gel and electrode buffers, as well as staining schedules, that provide clear starch gel enzyme banding for 22 enzyme systems in ferns. Requests for advice resulting from the recent surge of interest in fern enzyme electrophoresis have prompted us to compile our procedural data so that other researchers can take advantage of our experimentation. We hope that these data will stimulate more extensive electrophoretic investigation of pteridophytes and other electrophoretically difficult taxa. Gottlieb (1981b) recently reviewed aspects of enzyme electrophoresis primarily in gymnosperms and angiosperms. His discussion is equally relevant to understanding the potential applications and limitations of electrophoretic evidence in pteridophytes. Since homosporous pteridophytes have high chromosome numbers, it is tempting to invoke polyploidy in interpreting their enzyme band patterns. It is well

Starch Gel Electrophoresis of Ferns: A Compilation of Grinding Buffers, Gel and Electrode Buffers, and Staining Schedules

Gel electrophoresis of proteins has become a standard and powerful research tool for application in a multitude of biological disciplines One form of protein electrophoresis, isozyme analysis, has become particularly prominent in systematic and evolutionary biology as well as agronomy (Tanksley and Orton, 1983) Isozymes, or multiple molecular forms of enzymes, are enzymes that share a common substrate but differ in electrophoretic mobility (Markert and Moller, 1959) They are revealed when tissue extracts are subjected to electrophoresis in various types of gels and subsequently submersed in solutions containing enzyme-specific stains Genetic analysis may indicate that some of the variant electromorphs are encoded by alternate alleles at a single locus, in which case the allelic products are termed allozymes (Prakash et al, 1969) Data retrieved from electrophoretic gels consist of the number and relative mobilities of various enzyme products, which with appropriate genetic analyses become transformed into single or multilocus genotypes for each individual analyzed Reasons are many for the popularity of electrophoretic data (Avise, 1975; Gottlieb, 1977; Crawford, 1983), but foremost among these is that isozymes provide a series of readily scored, single-gene markers

Visualization and Interpretation of Plant Isozymes

The technique of starch gel electrophoresis of enzymes with specific staining for activity in the gel, the so-called zymogram method developed by Hunter and Markert (1957), has found many research applications. Screening studies, comparing relatively large numbers of enzymes among a variety of tissues and organisms, are finding increasing use in research on population genetics, taxonomy, etc. The methods presently available are scattered through the literature. This compilation is published in response to numerous requests from investigators in many fields. Most of the methods outlined here have been used primarily for tissue extracts from mammalian species. A few have been developed only on plants or lower animals, but this is not to say that they will not work on higher organisms. The buffer systems have been for the most part empirically arrived at. All are probably subject to improvement. The techniques of making the gels and carrying out the electrophoresis are not described here. These have been amply presented in a number of publications. The vertical system is essentially that described by Shaw and Koen (1968b). The horizontal system is essentially as described by Beckman and Johnson (1964). The horizontal method has the advantage of employing simpler and less expensive apparatus, and it can be run at room temperature using ice trays to keep the gels cold. For many enzyme systems, it provides as good results as the vertical. Both Connaught starch (Connaught Laboratories, Toronto) and Electrostarch (Otto Hiller Company, Madison, Wisconsin) will give satisfactory results with either technique. Unfortunately, different batches of starch may produce varying results, both in rate of migration and in resolution; also, some batches contain substances

Starch gel electrophoresis of enzymes—A compilation of recipes

The electrophoretic mobilities in starch gels have been determined for eight enzymes in extracts of representative cultures of members of the family Enterobacteriaceae. These values were compared with each other and with those obtained from certain bacteria not in this family. The migrations of the eight enzymes were virtually identical for each of eight strains of Escherichia coli and for two species of Shigella. A number of these enzymes appeared to be identical in other organisms believed to be closely related to E. coli (Salmonella), and the number of differences increased in organisms which appeared to have lesser degrees of relatedness by other criteria (deoxyribonucleic acid base compositions, overall similarity).

Zone Electrophoresis of Enzymes in Bacterial Taxonomy

α-Glycerophosphate dehydrogenase (GPDH) occurs in Drosophila melanogaster in three isozymic forms. These are separable by starch gel electrophoresis and have been tentatively numbered 1, 2, and 3. GPDH-1 is most concentrated in the adult thorax and GPDH-3 in the abdomen; 1 and 3 are in approximately equal amounts in the head. GPDH-2 is relatively weak in all preparations. In larvae, only GPDH-3 is present. Purified GPDH-1 has optimal activity at pH 6.7–7.0. GPDH-3 at pH 7.5, and GPDH-2 is intermediate. Changes in total GPDH activity parallel larval growth, pupal histolysis, and differentiation of adult tissues. In the latter period the ratio of activity at pH 6.7 to pH 7.6 increases, reflecting the shift from GPDH-3 to GPDH-1. Two types of homozygous GPDH patterns which differ in the electrophoretic mobilities of all three isozymes have been found in inbred strains. In heterozygous adults six bands, the parental forms of GPDH-1 and GPDH-3 and “hybrid” forms of each, can be resolved. Analysis of F2 and backcross progeny suggests that a single genetic locus affects all three isozymes. Heterozygous embryos have only the maternal form of GPDH-3 until just before they hatch as first instar larvae. At this stage they have maternal and paternal GPDH-3 plus an intermediate band.

Genetics and ontogeny of alpha-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster.

Degree of genetic similarity was estimated by electrophoretic comparison of 8–22 homologous proteins among several species of bacteria, slime molds, bony fishes, and bats. Genetic proximity generally varied directly with phylogenetic proximity, but even between closely related species more than half of the genetic loci were different. Although the times of divergence of the species studied are not known, it seems unlikely that this amount of genetic change could be effected entirely by selection. It also appears that many of the enzyme differences have little or no effect on activity. The results thus tend to support the position that a large part of evolutionary change is effected by random incorporation of selectively neutral mutations.

How many genes evolve

The activities of several enzymes were determined in homogenates of Drosophila melanogaster embryos and early larvae. The dehydrogenases of isocitrate (IDH), α-glycerophosphate (αGPDH), glucose 6-phosphate (G6PD), and 6-phosphogluconate (6PGD) show no increase in activity until late in the embryonic period (about 20 hr). The increase in the activity of these enzymes begins before hatching and continues into the larval period. Embryos heterozygous at loci controlling specific enzymes, IDH, αGPDH, G6PD, and ADH (alcohol dehydrogenase), have only the maternal form of each enzyme throughout most of the embryonic period. Paternal and hybrid forms of the enzymes are found at hatching. This corresponds to the increase in total activity for each of the enzymes. The results are discussed in terms of the control of gene expression in development.

Charles R. Shaw

Papers

Starch gel electrophoresis of enzymes—A compilation of recipes

Zone Electrophoresis of Enzymes in Bacterial Taxonomy

Genetics and ontogeny of alpha-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster.

How many genes evolve

Time of Expression of Genes Controlling Specific Enzymes in Drosophila Embryos