Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.     

Growth of Legionella pneumophila in association with blue-green algae (cyanobacteria).

Legionella pneumophila (Legionnaires disease bacterium) of serogroup 1 was isolated from an algal-bacterial mat community growing at 45 degrees C in a man-made thermal effluent. This isolate was grown in mineral salts medium at 45 degrees C in association with the blue-green alga (cyanobacterium) Fischerella sp. over a pH range of 6.9 to 7.6. L. pneumophila was apparently using algal extracellular products as its carbon and energy sources. These observations indicate that the temperature, pH, and nutritional requirements of L. pneumophila are not as stringent as those previously observed when cultured on complex media. This association between L. pneumophila and certain blue-green algae suggests an explanation for the apparent widespread distribution of the bacterium in nature.     

Increased recovery of salmonellae from environmental samples enriched with buffered peptone water.

The incidence and persistence of salmonellae in weather pools on the top of Stone Mountain were investigated with lactose and buffered peptone water used as pre-enrichment broths. A total of 162 samples were collected from 16 weather pools over a 3-month period. The use of buffered peptone water increased the recovery of salmonellae by approximately 25%. The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and pre-enrichment in buffered peptone water followed by enrichment in tetrathionate broth made it possible to detect all 37 of the contaminated samples. All of the isolates were Salmonella bareilly, the only serotype recovered in a previous study. All but one of the isolations were made from moist or wet samples. S. bareilly was isolated from rabbit dung and litter collected near the weather pools, but attempts to trap rabbits for study were unsuccessful. Random samples taken along a side of the mountain yielded S. bareilly in weather pools within the upper third of the mountain; below this level, S. weslaco and S. memphis were recovered, but not S. bareilly.     

Both ankyrin and band 4.1 are required to restrict the rotational mobility of band 3 in the human erythrocyte membrane.

A population of band 3 proteins in the human erythrocyte membrane is known to have restricted rotational mobility due to interaction with cytoskeletal proteins. We have further investigated the cause of this restriction by measuring the effects on band 3 rotational mobility of rebinding ankyrin and band 4.1 to ghosts stripped of these proteins as well as spectrin and actin. Rebinding either ankyrin or 4.1 alone has no detectable effect on band 3 mobility. Rebinding both these proteins together does, however, reimpose a restriction on band 3 rotation. The effect on band 3 rotational mobility of rebinding ankyrin and 4.1 are similar irrespective of whether or not band 4.2 is removed from the membrane. We suggest that ankyrin and 4.1 together promote the formation of slowly rotating clusters of band 3.     

Ecological distribution of Legionella pneumophila.

Bacteria were concentrated 500-fold from 20-liter water samples collected from 67 different lakes and rivers in the United States. The data suggest that Legionella pneumophila is part of the natural aquatic environment and that the bacterium is capable of surviving extreme ranges of environmental conditions. The data further demonstrate the effectiveness of the direct fluorescent-antibody technique for detecting L. pneumophila in natural aquatic systems. Smears of the concentrated samples were screened microscopically for serogroups of L. pneumophila by the direct fluorescent-antibody technique. Virtually all of the 793 samples were found to be positive by this method. The 318 samples containing the largest numbers of positive bacteria which were morphologically consistent with L. pneumophila were injected into guinea pigs for attempted isolations. Isolates were obtained from habitats with a wide range of physical, chemical, and biological parameters. Samples collected monthly from a thermally altered lake and injected into guinea pigs demonstrated a seasonality of infection, with the highest frequency of infection occurring during the summer months.     

Protein-protein interactions of cytochrome oxidase in inner mitochondrial membranes. The effect of liposome fusion on protein rotational mobility

Rotational diffusion of cytochrome oxidase in the inner membrane of rat liver mitochondria was measured by detecting the decay of absorption anisotropy after photolysis of the heme a3.CO complex by a vertically polarized laser flash. As in previous experiments with beef heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510), co-existence of rotating cytochrome oxidase (mean rotational relaxation time, phi, of 700 to 1400 microseconds) and immobilized cytochrome oxidase (phi greater than 20 ms) was observed in mitochondria and mitoplasts. The effect of lipid/protein ratio by weight (L/P) on the relative proportions of mobile and immobile cytochrome oxidase was investigated following the fusion of soybean phospholipid vesicles with mitoplasts. The fusion procedure yielded four separate fractions upon sucrose density gradient centrifugation with L/P as follows: 0.3 in Pellet, 0.7 in Band 3, 1.5 in Band 2, and 3.0 in Band 1. The percentage of rotationally mobile cytochrome oxidase (phi = 700 to 1000 microseconds) in each of the different bands was found to be 16% in Pellet, 25% in Band 3, 47% in Band 2, and 76% in Band 1 at 37 degrees C. The dependence of the amount of mobile cytochrome oxidase on L/P indicates that the fraction of aggregated protein progressively decreases with decreasing concentration of proteins in the membrane. Thus, the large immobile fraction of cytochrome oxidase in mitochondrial inner membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P. The decrease in the mobile fraction in Pellet compared with mitoplasts was shown to be due to the pH 6.5 incubation used for fusion.     

Genetics of lipid deposition in the Japanese quail

Summary The research presented here was designed to investigate the mode of inheritance of fat and lean tissue deposition, and the relationship between them and body weight in Japanese quail. Heterotic effects were found for weight, size, and number of adipocytes in the abdominal fat depots, weight of the sartorial fat depot and percentage carcass fat with means for the hybrids being lower than those for the parental lines. General inferences concerning the importance of nonadditive genetic variation for lean and body weight were precluded due to inconsistencies observed among mating combinations. Thus, although heterosis and recombination effects were general for characteristics associated with fat deposition, the situation for body weight and lean was unique to the populations involved. It may be hypothesized that heterosis in the efficiency of feed utilization is reflected by the heterosis for fat deposition which explains why hybrids utilize feed better than their parental lines.     

Variations in population levels of citrus blackfly,Aleurocanthus woglumi [Hom: Aleyrodidae] and parasites during an eradication program in Florida

From November, 1976 until October, 1979 citrus blackfly,Aleurocanthus woglumi Ashby and its parasite populations were sampled monthly in urban areas of Broward County, Florida. During this period, the parasites,Amitus hesperidum Silvestri andProspaltella opulenta Silvestri became established and exerted control onA. woglumi populations. Parasite succession was noted in thatA. hesperidum was first abundant and later replaced in abundance byP. opulenta. The 2 parasite species continued to coexist and exert mortality on low pest populations in spite of chemical spraying used for eradication showing that integrated control ofA. woglumi is a definite possibility now that the pest is well established in Florida.

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Résumé De novembre 1976 à octobre 1979, on a échantillonné mensuellement dans les zones urbaines du Comté de Broward, Floride, les populations de la mouche noire des citrus,Aleurocanthus woglumi Ashby et de ses parasites. Pendant cette période les parasites,Amitus hesperidum Silvestri etProspaltella opulenta Silvestri se sont établis et ont limité les populations deA. woglumi. Une succession dans les parasites a été notée:A. hesperidum fut abondant d'abord et remplacé ensuite en fréquence parP. opulenta. Les 2 espèces ont continué à coexister et causent la mortalité des faibles populations du ravageur malgré les traitements chimiques utilisés pour l'éradication; ce qui montre la possibilité d'une lutte intégrée contreA. woglumi maintenant que ce ravageur est bien établi en Floride.

     

The influence of coal ash and thermal discharges upon the distribution and bioaccumulation of aquatic invertebrates

The distribution, density and uptake of twenty elements by aquatic invertebrates inhabiting a drainage system, that received excessive coal ash effluent (275 JTU of turbidity) at one end and thermal loading (44.5°C) at the other end, was studied for 15 months. The ash settling basin filled during the first eight months of sampling which resulted in the release of ash effluent directly into the receiving system. Density of invertebrates was lowest in the 300 m stream between the ash basin and swamp and highest 1200 m beyond the stream-swamp confluence where ash influence was minimal. Invertebrate density was lowest in the stations where turbidity from ash effluent was greatest. The most tolerant invertebrates to coal ash stress were odonates (Libellula sp. and Enallagma sp.), crayfish (Procambarus sp.), amphipods (Gammarus sp.) and gastropods (Physa. sp.), and midges (Chironomidae) when the basin was filling. During the period of ash overflow, all groups were either reduced in numbers or absent. In the thermally stressed station, Libellula sp. was the predominant invertebrate sampled when water temperature ranged from 25.5–45.5°C (257-1=28.7°C) all aquatic invertebrates were limited in numbers and density when temperature exceeded the lower and upper ranges of 10.0–38.0°C.This research was supported by AEC Contract AT (38-1-824)This research was supported by AEC Contract AT (38-1-824)     

Lethal temperatures of citrus blackflyAleurocanthus woglumi [Hom.: Aleyrodidae] and its parasite,Amitus hesperidum [Hym.: Platygasteridae]

Seasonally acclimatized citrus blackfly (CBF),Aleurocanthus woglumi Ashby, and its parasite,Amitus hesperidum Silv. were exposed to extreme temperatures for 3-hour periods and survivorship measured. In summer experiments, the LD₅₀ for CBF adults and 1st instar nymphs occurred between 40° and 45°C. The LD₅₀ forA. hesperidum adults occurred between 35° and 40°C and increased with decreased humidity, although some adults could continue to emerge from parasitized CBF up to 45°C. In winter experiments, the LD₅₀ for CBF eggs and adults occurred between ?5° and ?10°C and for other CBF stages between ?10° and ?15°C. The LD₅₀ for adultA. hesperidum, and survival and emergence of the parasites from CBF both occurred between ?10° and ?15°C. Comparing data from the 2 seasons shows that adults are the most temperature sensitive stage of CBF and the parasite is more temperature sensitive than CBF, especially at higher temperatures. Correlating lethal temperature data with field meteorological conditions shows that short-term temperature exposures cannot be expected to stop the potential spread of CBF orA. hesperidum through Florida.