Continuous Free-Film Electrophoresis: The Crescent Phenomenon

Electroosmotic and laminar flow effects in continuous free-film electrophoresis cause the separated bands to assume crescent-shaped cross-sections. These are made visible by a special illumination technique. The crescent characteristics – their direction and amplitude of curvature, symmetry, and extension in depth – provide a “signature” of separation performance. This permits the operator to adjust the cell wall zeta potential for optimum resolution, to increase the useful level of sample flow rate, and to recognize and correct causes of instability.     

3 beta-Hydroxysteroid dehydrogenase in human placental microsomes and mitochondria: co-solubilization of androstene and pregnene activities

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) was solubilized from human term-placental microsomes and mitochondria using the non-ionic detergent, polyoxyethylene 20 cetyl ether (BrijR-58). Electron photomicrographs showed microsomes and mitochondria well disrupted by the detergent. The pregnene (C-21) and androstene (C-19) activities co-solubilized over a range (0.04-0.44) of BrijR-58/protein (B/P) concentration ratios (w/w). Optimal solubilization of the C-19 and C-21 activities were 63.3 +/- 2.6% (mean +/- SEM) from mitochondria (B/P ratio 0.37) and 71.8 +/- 2.1% from microsomes (B/P ratio 0.34). Detergent treatment of microsomes and mitochondria--varying time (5-90 min, pH 7.4) or varying pH (6.0-7.8, 90 min)--yielded C-19 activities identical with C-21 activities. The C-21/C-19 specific activity ratios of 3 beta-HSD in particulate, solubilized and chromatographed preparations were 2.28 +/- 0.16 (mean +/- SEM) for mitochondria and 1.97 +/- 0.07 for microsomes. 3 beta-HSD molecular weight estimates were 208,000 (microsomes) and 220,000 (mitochondria). These studies argue that a single protein is responsible for both the C-19 and C-21 activities of 3 beta-HSD and that this protein is the same in microsomes and mitochondria.     

Contrast in levels of metabolic enzymes in human and mouse ova

A methodology is described for analyzing single human ova for 8 or 9 different metabolic enzymes, or 4 or 5 enzymes plus as many metabolites. This overcomes an obstacle to the study of human ovum metabolism: the severe limitation of usable material. Results obtained with this methodology, applied to discarded specimens from an in vitro fertilization program, indicate that in spite of imperfections these ova can provide a valid picture of the metabolic characteristics of normal human ova. Data are presented for 17 enzymes from 8 metabolic pathways in human and mouse ova. Relative to size, 10 of the enzymes were substantially higher in human than mouse ova. Most dramatically so were 2 enzymes of fatty acid metabolism (10-fold and 15-fold), hexokinase (9-fold), and aspartate aminotransferase (19-fold). This suggests that major species differences in metabolism are present. The validity of the human data, in spite of restriction to discarded material, is supported by (1) consistency of results among most of the ova, 2] concordance between average levels with those of rare specimens that were discarded because sperm were not available, and (3) the presence of adenosine triphosphate (ATP) concentrations similar to those of normal mouse ova. Surprisingly, both human and mouse ova contain phosphocreatine at levels nearly equal of those of ATP.     

CFM1, a member of the CRM-domain protein family,functions in chloroplast group II intron splicing in Setaria viridis

The chloroplast RNA splicing and ribosome maturation (CRM) domain is a RNA-binding domain found in a plant-specific protein family whose characterized members play essential roles in splicing group I and group II introns in mitochondria and chloroplasts. Together, these proteins are required for splicing of the majority of the approximately 20 chloroplast introns in land plants. Here, we provide evidence from Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. A Setaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs: trnI, trnV and trnA. Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole-plant chlorotic phenotypes. Co-immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in the cfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.