HETEROGENEOUS DISTRIBUTION OF ENZYMES IN SUBMICROSOMAL MEMBRANE FRAGMENTS

Microsomal membranes are postulated to contain either a homogeneous arrangement of individual enzymes or groupings of functionally related enzymes. In the present study we attempt to distinguish between these hypotheses in subfractions of rough microsomes from rat liver. After sonication, the individual vesicles that make up the rough-membrane fraction average less than 1/100 of their previous mass. The vesicles in the sonicated suspension are fractionated roughly according to size on a continuous sucrose gradient. Enzyme activity or concentration in fractions of the gradient is expressed on a phospholipid basis. Fractions containing primarily small vesicles differ from those containing larger vesicles in a manner suggesting a certain degree of separation of NADH-linked from NADPH-linked enzymes. NADH-ferricyanide reductase, NADH-cytochrome c reductase and cytochrome b₅ are most concentrated within the large vesicles in the lowest third of the gradient. In contrast, NADPH-cytochrome c reductase and cytochrome P-450 are found in highest concentration in the small vesicles that make up the upper third of the gradient. The results suggest a nonrandom distribution of these two enzyme groups in the membranes of the endoplasmic reticulum.     

Replication of nucleolus-associated DNA during "G2 phase" in Physarum polycephalum

In the myxomycete, Physarum polycephalum, the bulk of nuclear DNA replication occurs during a period of a few hours immediately following upon mitosis. During the remainder of the intermitotic period, incorporation of thymidine-³H continues at a low rate in the region of the nucleolus (radioautographs). A few nuclei incorporated thymidine-³H into the extranucleolar chromatin at a high rate at all times of the intermitotic period. These nuclei were exceptionally large and they frequently contained several small nucleoli of different sizes rather than the one, central nucleolus which is characteristic of a normal interphase nucleus.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Book Reviews

Book reviewed in this article:
LINGUISTICS: Pis'mennost'

     

Elemental composition and food intake of Phialidium hydromedusae in the laboratory

Elemental composition and feeding rate of hydromedusae Phialidium sp. on copepods were studied in the laboratory. Regression equations for both mature and immature medusae allowed the estimation of their dry weight (DW), total C and N content as a function of their diameter. The mean C content as percentage of the DW varied from 13.13% ( ) for the immature to 19.38% (5.68) for the mature individuals. The mean N content is 4.03% (2.49) of DW of immatures and 5.85% (2.70) of the matures. Ingestion rate of Phialidium sp. fed on copepods (200–500 μm) increased with prey density but reached a maximum at high prey concentrations. A maximum ingestion rate of 8.55 (1.6) copepods · medusa ⁻¹ · h⁻¹ was reached for prey concentrations of > 140 copepods · 1 ⁻¹ for both immature and mature medusae. Maximum daily consumption of prey weight varied from 1.41 to 978% C body weight for mature medusae and from 2.90 to 975% for the immature individuals.     

Involvement of the Dihydropyridine Receptor and Internal CaStores in Myoblast Fusion

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca²⁺associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca²⁺is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca²⁺but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca²⁺channel) and the internal stores of Ca²⁺. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca²⁺is triggered by the type of mechanism already described in skeletal muscle.     

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA⁺)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.     

Effect of enhanced UV-B radiation on pollen quantity,quality, and seed yield in Brassica rapa (Brassicaceae)

We conducted three experiments to examine the influence of ultraviolet-B radiation (UV-B; 280–320 nm) exposure on reproduction in Brassica rapa (Brassicaceae). Plants were grown in a greenhouse under three biologically effective UV-B levels that simulated either an ambient stratospheric ozone level (control), 16% (“low enhanced”), or 32% (“high enhanced”) ozone depletion levels at Morgantown, WV, USA in mid-March. In the first experiment, we examined whether UV-B level during plant growth influenced in vivo pollen production and viability, and flower production. Pollen production and viability per flower were reduced by ≈50% under both enhanced UV-B levels relative to ambient controls. While plants under high-enhanced UV-B produced over 40% more flowers than plants under the two lower UV-B treatments, whole-plant production of viable pollen was reduced under high-enhanced UV-B to 17% of that of ambient controls. Whole-plant production of viable pollen was reduced under low-enhanced UV-B to 34% of ambient controls. In the second experiment, we collected pollen from plants under the three UV-B levels and examined whether source-plant UV-B exposure influenced in vitro pollen germination and viability. Pollen from plants under both enhanced-UV-B treatments had initially lower germination and viability than pollen from the ambient level. After in vitro exposure to the high-enhanced UV-B levels for 6 h, viability of the pollen from plants grown under ambient UV-B was reduced from 65 to 18%. In contrast, viability of the pollen from plants grown under both enhanced UV-B treatments was reduced to a much lesser extent: only from ≈43 to 22%. Thus, ambient source-plant pollen was more sensitive to enhanced UV-B exposure. In the third experiment, we used pollen collected from source plants under the three UV-B levels to fertilize plants growing under ambient-UV-B levels, and assessed subsequent seed production and germination. Seed abortion rates were higher in plants pollinated with pollen from the enhanced UV-B treatments, than from ambient UV-B. Despite this, seed yield (number and mass) per plant was similar, regardless of the UV-B exposure of their pollen source. Our findings demonstrate that enhanced UV-B levels associated with springtime ozone depletion events have the capacity to substantially reduce viable pollen production, and could ultimately reduce reproductive success of B. rapa.