Reconstitution of the two terminal enzymes of the heme biosynthetic pathway into phospholipid vesicles

Purified mouse protoporphyrinogen oxidase (EC 1.3.3.4) and ferrochelatase (EC 4.99.1.1), the two terminal enzymes of the heme biosynthetic pathway, have been reconstituted into phospholipid vesicles, and the kinetics of the enzymes in the reconstituted systems were compared with the values obtained with the free enzymes. The apparent Km for free protoporphyrinogen oxidase in detergent solution is 5.61 +/- 0.62 microM for free protoporphyrinogen. The Km was lower when the enzyme was inserted into phospholipid vesicles (0.78 +/- 0.28 microM) and when both enzyme and substrate were incorporated into phospholipid vesicles (0.61 +/- 0.14 microM). In the presence of cardiolipin, a phospholipid present mainly in the inner mitochondrial membrane, the value of the Km for the substrate decreased 3-fold (0.20 +/- 0.02 microM). For reconstituted ferrochelatase similar kinetic analyses were carried out and it was found that the apparent Km values were only weakly affected by the lipid environment. Studies on the orientation of ferrochelatase demonstrated that approximately 50% of the enzyme in the reconstituted system had the active site located in the inner face of the phospholipid vesicle. This is in contrast to intact mitochondria where the active site is located on the matrix side of the inner mitochondrial membrane. The activation energies for both enzymes were determined for free and reconstituted enzymes. It was found that for both enzymes the activation energies were lower for the reconstituted systems than for the free enzymes.     

Differential response to environmental alcohol among second-chromosome arrangements in experimental populations of Drosophila buzzatii

Drosophila buzzatii feeds and breeds on the decaying cladodes and fruits of several species of Opuntia (prickly pear) which contain significant levels of ethanol and isopropanol. The potential influence of these two alcohols on the inversion polymorphism of the second and fourth chromosomes was investigated in ten experimental populations with different amounts of alcohol (either ethanol or isopropanol) added to the culture medium. All populations were started with the offspring of 29 wild females collected at Adeje (Tenerife, Canary Islands) and their genetic composition was monitored for about two years (more than 30 generations). Consistent changes in the frequency of most second- and fourth-chromosome arrangements occurred in all populations including those without alcohol (control). The comparison of inversion frequency through the various treatments revealed a significant influence of the alcohol on the frequency changes of the four second-chromosome arrangements. Moreover, this influence was of a different type for every one of them, sometimes with opposite effects between alcohols and/or concentrations. These results indicate genetic differentiation among second-chromosome arrangements with regard to alcohol and suggest that the alcohol heterogeneity found in the species' trophic niche may play an important role in the maintenance of this polymorphism and also in the recent historical changes in the frequency of some arrangements associated with colonization.This paper is number X of the series The evolutionary history of Drosophila buzzatii.     

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and Bundle Sheath Chloroplasts of Maize

Distribution of the major light-harvesting chlorophyll a/b-protein (LHCII) and its mRNA within bundle sheath and mesophyll cells of maize (Zea mays L.) was studied using in situ immunolocalization and hybridization, respectively. In situ hybridization with specific LHCII RNA probes from maize and Lemna gibba definitively shows the presence of high levels of mRNA for LHCII in both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, demonstrate the presence of LHCII polypeptides in chloroplasts of both cell types. The polypeptide composition of LHCII and the amount of LHCII in bundle sheath cells are different from those in mesophyll cells. Both mesophyll and bundle sheath chloroplasts can take up, import and process the in vitro transcribed and translated LHCII precursor protein from L. gibba. Although bundle sheath chloroplasts incorporate LHCII into the pigmented light-harvesting complex, the efficiency is lower than that in mesophyll chloroplasts.     

The effect of glucose-6-phosphate isomerase genotype onin vitro specific activity andin vivo flux inMytilus edulis

Four samples of the musselMytilus edulis were taken between 1984 and 1987 from Stony Brook, New York, and used to study the glucose-6-phosphate isomerase (GPI) polymorphism in this species.In vitro specific activity andin vivo flux measured in the same animals were found to be significantly correlated. A significant effect of GPI genotype on flux was observed in one of the samples; overall, significant evidence of effect of genotype on enzyme activity was also obtained. GPI activities of common genotypes tend to deviate less from the population mean than those of rare (frequency less than 5%) genotypes. This suggests the possibility that rare GPI genotypes are rare as a consequence of having biochemical properties that deviate from an optimum level and, therefore, having a lower fitness. In support of this hypothesis, we found in one of our samples that shell length is a concave function of GPI activity with an intermediate optimum activity level. The financial support provided to P.J.N.S. by the Luso-American Educational Commission (Fulbright Program), the Instituto Nacional de Investigacao Científica (Portugal), and the Faculdade de Ciências da Universidade de Lisboa during several stages of this research is gratefully acknowledged. Financial support from the Ministerio de Educatión y Ciencia (Spain) in the form of a postdoctoral Fulbright/MEC fellowship to M.S. is also gratefully acknowledged. Research was supported by National Science Foundation Grant BSR-8415060 to R.K.K. This is contribution No. 736 from the Program in Ecology and Evolution, State University of New York at Stony Brook. On leave from Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande C2, Lisboa, Portugal.     

Prevention of insulin resistance by environmental manipulation as young rats mature

This study was initiated in an attempt to see if the insulin resistance associated with maturation in young rats could be prevented by environmental manipulation. Consequently, seven week-old rats were either housed in standard laboratory cages and fed a calorie-restricted diet or placed individually in exercise wheel cages and allowed to eat chow ad lib. A control group of rats was housed in standard laboratory cages from seven weeks to five months of age, and also allowed to eat chow ad lib. When studied at five months of age, the chow-fed rats weighed more (624 +/- 8 g) than either the calorie restricted (479 +/- 9 g) or exercise trained (485 +/- 13 g) rats. Insulin action was compared in the three groups by assessing the steady-state serum glucose (SSSG) and insulin (SSSI) concentrations achieved during a continuous intravenous infusion of glucose and exogenous insulin. The results of these studies indicated that SSSG concentration was significantly higher (P less than 0.001) in chow-fed rats than in the two experimental groups. Since SSSI concentrations were the same in all three groups, lower SSSG concentrations in calorie-restricted and exercise trained rats indicates that insulin-stimulated glucose uptake was preserved in these two groups as compared to the chow-fed population. In an attempt to understand why exercise training and calorie restriction prevented the development of insulin resistance, muscle glycogen synthase activity and muscle capillary density were compared in the three groups of five month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cytotaxonomy ofEmilia spp. (Asteraceae: Senecioneae) occurring in Brazil

Analysis of several populations in a large part of the distribution area of the genusEmilia in Brazil has revealed only two species: the diploidE. sonchifolia and the tetraploidE. fosbergii. The more widely reportedE. coccinea was not found. They show a karyotype constancy in morphology and chromosome number (2n = 10 and 2n = 20, respectively), C-banding pattern and number of secondary constrictions. Some indications were found thatE. fosbergii may be an allopolyploid and that its ancestors had different genome sizes.     

Mouse protoporphyrinogen oxidase. Kinetic parameters and demonstration of inhibition by bilirubin.

The penultimate step of haem biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, was examined with purified murine hepatic protoporphyrinogen oxidase (EC 1.3.3.4) in detergent solution. The kinetic parameters for the two-substrate (protoporphyrinogen and oxygen) reaction were determined. The limiting Km for protoporphyrinogen when oxygen is saturating is 6.6 microM, whereas the Km for oxygen with saturating concentrations of protoporphyrinogen is 125 microM. The kcat. for the overall reaction is 447 h-1. The ratio of kcat. to the Km for protoporphyrinogen is approx. 20-fold greater than the kcat./Km,O2 ratio. The ratio of protoporphyrin formed to dioxygen consumed is 1:3. Ubiquinone-6, ubiquinone-10 and dicoumarol stimulate protoporphyrinogen oxidase activity at low concentrations (less than 15 microM), whereas coenzyme Q0 and menadione show no activation at these concentrations. Above 30 microM, all five quinones inhibit the enzyme activity. FAD does not significantly affect the activity of the enzyme. Bilirubin, a product of haem catabolism, is shown to be a competitive inhibitor of the penultimate enzyme of the haem-biosynthetic pathway, protoporphyrinogen oxidase, with a calculated Ki of 25 microM. The terminal enzyme of haem-biosynthetic pathway, namely ferrochelatase, is not inhibited by bilirubin at concentrations over double the Ki value for the oxidase. In contrast with other enzymic systems, the toxicity of bilirubin is not reversed by binding to albumin.     

Molecular analysis of yellow fever virus 17DD vaccine strain.

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared worldwide. As part of a broader approach to determine the genetic variability in YF 17D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purified directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF 17D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot hybridization of virion RNAs of purified 17DD with two other strains of YF 17D virus revealed only genome-sized molecules for all three viruses. These observations suggest that the vaccine phenotype is primarily associated with the accumulation of mutations.