Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.     

The human glutathione S-transferase. Studies on the kinetic, stability and inhibition characteristics of the erythrocyte enzyme

Bromosulphophthalein and N-ethylmaleimide, inhibitors of glutathione S-transferase (EC 2.5.1.18 RX: glutathione R. transferase), have been used to identify variant forms of the erythrocyte enzyme. One 'atypical' sample was detected and was shown to have appreciably different kinetic and stability properties. These inhibitors may be useful in surveys of variation in this group of enzymes.     

Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase.

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.     

Circadian rhythms in the activity of a plant protein kinase.

Bryophyllum fedtschenkoi is a Crassulacean acid metabolism plant whose phosphoenolpyruvate carboxylase is regulated by reversible phosphorylation in response to a circadian rhythm. A partially purified protein kinase phosphorylated phosphoenolpyruvate carboxylase in vitro with a stoichiometry approaching one per subunit and caused a concomitant 5- to 10-fold decrease in the sensitivity of the carboxylase to inhibition by malate. The sites phosphorylated in vitro were identical to those phosphorylated in intact tissue. The activity of the protein kinase was controlled in a circadian fashion. During normal diurnal cycles, kinase activity appeared between 4 and 5 h after the onset of darkness and disappeared 2----3 h before the end of darkness. Kinase activity displayed circadian oscillations in constant environmental conditions. The activity of protein phosphatase 2A, which dephosphorylates phosphoenolpyruvate carboxylase, did not oscillate. Treatment of detached leaves with the protein synthesis inhibitors puromycin and cycloheximide blocked the nocturnal appearance of the protein kinase activity, maintained phosphoenolypyruvate carboxylase in the dephosphorylated state and blocked the circadian rhythms of CO2 output that is observed in constant darkness and CO2-free air. The simplest explanation of the data is that there is a circadian rhythm in the synthesis of phosphoenolpyruvate carboxylase kinase.     

The inheritance of skeletal muscle fibre composition in mice

Two inbred strains of mice C3H/HE and SWR, and generations produced by intercrossing, were studied to investigate the effect of heredity on muscle composition. The data were found to be consistent with a polygenic mode of inheritance. No simple effect of sex-linkage or maternal influence was evident. The heritability of fibre type percentage total fibre number and the relative size of Type I and Type II fibres were highly significant. Principal component analysis yielded a "genetic" vector which accounted for 57% of the variation in muscle fibre composition. Patterns of covariance of fibre type percentage, total fibre number and relative sizes of Type I and Type II fibres showed a single correlated response.     

A comparison of two methods for fitting the integrated Michaelis–Menten equation (Short Communication)

The methods of Atkins & Nimmo (1973) and Fernley (1974) for fitting the integrated Michaelis-Menten equation were compared by using the same sets of simulated experimental data. The method of Fernley (1974) is to be preferred because it gives precise and unbiased estimates of the Michaelis-Menten parameters over a wide range of substrate concentrations. However, the estimates may not be symmetrically distributed, especially at low substrate concentrations.     

Rhythms in magnesium ion inhibition and hysteretic properties of nitrate reductase in the CAM plant Bryophyllum fedtschenkoi

Nitrate reductase (NR, EC 1.6.6.1) was tested in crude extracts of leaves from Bryophyllum fedtschenkoi plants growing under alternating light/darkness as well as in excised leaves kept in continuous light or darkness. In most extracts NR activity was inhibited 20–80% by 5 m M Mg²⁺ A light or darkness shift (30 min darkness) during the first part of the photoperiod gave an increase in the Mg²⁺ inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variations. Strongest inhibition was found in extracts made during the latter part of the photoperiod and start of the dark period. Pre-incubation of crude extracts with ATP increased Mg²⁺ inhibition, indicating that phosphorylation of NR is involved in regulation of NR in Crassulacean acid metabolism (CAM) plants. In continuous light an increase in Mg²⁺ inhibition occurred after 20 h and 40 h, indicating a rhythm in the phosphorylation of NR. A delay in the production of nitrite in the assay (hysteresis) was generally seen in extracts susceptible to Mg²⁺ inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in CO₂ exchange. However, in contrast to the rhythm in CO₂ exchange, NR rhythms were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that post-translational modification of CAM NR is influenced by light/darkness and by an endogenous rhythm.     

Regulation of nitrate reductase and phosphoenolpyruvate carboxylase activities in barley leaf protoplasts

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg²⁺ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO ₃ ^- ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO ₃ ^- . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - DMO 5,5-dimethyl-2,4 oxazolidinedione - NR nitrate reductase - PEPCase Phosphoenolpyruvate carboxylase This work was supported by the Norwegian Research Council by a Grant to C.L: L.H.S. was supported by the Biotechnology and Biological Sciences Research Council.     

Identifying historical and future global change drivers that place species recovery at risk

Ecosystem management in the face of global change requires understanding how co-occurring threats affect species and communities. Such an understanding allows for effective management strategies to be identified and implemented. An important component of this is differentiating between factors that are within (e.g. invasive predators) or outside (e.g. drought, large wildfires) of a local manager's control. In the global biodiversity hotspot of south-western Australia, small- and medium-sized mammal species are severely affected by anthropogenic threats and environmental disturbances, including invasive predators, fire, and declining rainfall. However, the relative importance of different drivers has not been quantified. We used data from a long-term monitoring program to fit Bayesian state-space models that estimated spatial and temporal changes in the relative abundance of four threatened mammal species: the woylie (Bettongia penicillata), chuditch (Dasyurus geoffroii), koomal (Trichosurus vulpecula) and quenda (Isoodon fusciventor). We then use Bayesian structural equation modelling to identify the direct and indirect drivers of population changes, and scenario analysis to forecast population responses to future environmental change. We found that habitat loss or conversion and reduced primary productivity (caused by rainfall declines) had greater effects on species' spatial and temporal population change than the range of fire and invasive predator (the red fox Vulpes vulpes) management actions observed in the study area. Scenario analysis revealed that a greater extent of severe fire and further rainfall declines predicted under climate change, operating in concert are likely to further reduce the abundance of these species, but may be mitigated partially by invasive predator control. Considering both historical and future drivers of population change is necessary to identify the factors that risk species recovery. Given that both anthropogenic pressures and environmental disturbances can undermine conservation efforts, managers must consider how the relative benefit of conservation actions will be shaped by ongoing global change.