Richard E. McCarty

Ferrous ion transport across chloroplast inner envelope membranes

The initial rate of Fe2+ movement across the inner envelope membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membrane vesicles loaded with the Fe2+-sensitive fluorophore, Phen Green SK. The rate of Fe2+ transport was rapid, coming to equilibrium within 3s. The maximal rate and concentration dependence of Fe2+ transport in predominantly right-side-out vesicles were nearly equivalent to those measured in largely inside-out vesicles. Fe2+ transport was stimulated by an inwardly directed electrochemical proton gradient across right-side-out vesicles, an effect that was diminished by the addition of valinomycin in the presence of K+. Fe2+ transport was inhibited by Zn2+, in a competitive manner, as well as by Cu2+ and Mn2+. These results indicate that inward-directed Fe2+ transport across the chloroplast inner envelope occurs by a potential-stimulated uniport mechanism.

Nitrite transport in chloroplast inner envelope vesicles. I. Direct measurement of proton-linked transport

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.

Molecular Dissection of the [epsilon] Subunit of the Chloroplast ATP Synthase of Spinach

The gene encoding the [epsilon] subunit (atpE) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia coli. The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain [epsilon] that is as biologically active as [epsilon] purified from chloroplast-coupling factor 1 (CF1). Recombinant [epsilon] folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF1 deficient in [epsilon] and restores proton impermeability to thylakoid membranes reconstituted with CF1 deficient in [epsilon]. Site-directed mutagenesis was used to generate truncations and single amino acid substitutions in the primary structure of [epsilon]. In the five mutants tested, alterations that weaken ATPase inhibition by recombinant [epsilon] affect its ability to restore proton impermeability to a similar extent, with one exception. Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeabilty. As in the case of E. coli, it appears that N-terminal truncations of the [epsilon] subunit have more profound effects than C-terminal deletions on the function of [epsilon]. Recombinant [epsilon] with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the [epsilon] of spinach and the [epsilon] of Pisum sativum, inhibits ATPase activity with a reduced potency similar to that of purified pea [epsilon]. Four of the six amino acids are serine or threonine. These hydroxylated amino acids may be important in [epsilon]-CF1 interactions.

Patterns of parental care in two cricetid rodents, Onychomys torridus and Peromyscus leucopus

Daily observations were made of the maternal and paternal behaviour of Onychomys torridus and Peromyscus leucopus in laboratory cages. In both species, the females spent more time in the nest area with the young and groomed the young more frequently than their male partners, but the males played a surprisingly active role in nest attendance. Paternal nest attendance was more frequent in O. torridus males than in P. leucopus males. Onychomys males were also more active in grooming the young than Peromyscus males. These species-specific patterns of parental care are discussed in relation to the behavioural and ecological characteristics of Onychomys and Peromyscus.

Copper Transport Across Pea Thylakoid Membranes

The initial rate of Cu2+ movement across the thylakoid membrane of pea (Pisum sativum) chloroplasts was directly measured by stopped-flow spectrofluorometry using membranes loaded with the Cu2+-sensitive fluorophore Phen Green SK. Cu2+ transport was rapid, reaching completion within 0.5 s. The initial rate of uptake was dependent upon Cu2+ concentration and saturated at about 0.6 μm total Cu2+. Cu2+ uptake was maximal at a thylakoid lumen pH of 7.0. Cu2+ transport was inhibited by Zn2+ but was largely unaffected by Mn2+ and Cu+. Zn2+ inhibited Cu2+ transport to a maximum of 60%, indicating that there may be more than one transporter for copper in pea thylakoid membranes.

Direct Measurement of Nitrite Transport Across Erythrocyte Membrane Vesicles Using the Fluorescent Probe, 6-Methoxy-N-(3-sulfopropyl) quinolinium

Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO2 in a pH-dependent manner. By contrast, erythrocyte ghosts had very low diffusion rates for nitrous acid. Erythrocyte ghosts preloaded with 50 mM nitrite to quench SPQ fluorescence were utilized to study heteroexchange with externally added anions. SPQ fluorescence increases (becomes unquenched) with added bicarbonate and nitrate, indicating that nitrite is moving out of the preloaded vesicles. The pH optimum for this exchange was approximately 7.6 and exchange was inhibited by N-ethylmaleimide (NEM) and dihydro-4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). These data indicate that nitrite moves across erythrocyte plasma membranes as NO2- by a heteroexchange mechanism with other monovalent anions.

Cross-species fostering: effects on the olfactory preference of Onychomys torridus and Peromyscus leucopus.

Litters of southern grasshopper mice (Onychomys torridus) and white-footed mice (Peromyscus leucopus) were reared by the natural parents or were reciprocally cross-fostered to parents of the opposite species. After weaning, mice were allowed a choice between the soiled beddings of Onychomys and Peromyscus adults of the opposite sex. Compared to controls, cross-fostered mice of both species displayed decreased preferences for homospecific odors. Cross-fostered Peromyscus males actually switched species preferences to Onychomys odors.

Regulation of proton flow and ATP synthesis in chloroplasts.

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 μmol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the γ and ε subunits of CF1 play key roles in both regulation of activity and proton translocation.

Differences between Two Tight ADP Binding Sites of the Chloroplast Coupling Factor 1 and Their Effects on ATPase Activity

Purified chloroplast ATP synthase (CF1) contains 1.2-2 mol of tightly bound ADP/mol of enzyme that resists removal by gel filtration or dialysis. CF1 was depleted of its endogenous nucleotide by treatment with alkaline phosphatase. Tightly bound nucleotide was demonstrated not to have an essential structural role. CF1 depleted of endogenous nucleotide retains its ability to catalyze Ca2+- and Mg2+-dependent ATPase activity and is not more sensitive to cold inactivation than untreated CF1. 2′(3′)-O-Trinitrophenyladenosine 5′-diphosphate (TNP-ADP) binds tightly to two sites on nucleotide-depleted CF1, binding to either site at a faster rate than that of exchange of bound nucleotide for medium nucleotide. The nucleotide-depleted enzyme binds about one additional mol of TNP-ADP/mol of CF1, indicating that there is a tight TNP-ADP binding site that does not exchange readily with medium nucleotide. It is MgADP in this nonexchanging site, not the easily exchanging ADP binding site, that is responsible for the MgADP-induced inhibition of the ATPase activity. The rate of exchange of tightly bound ADP from CF1 matches the rate at which the Mg2+ATPase activity of CF1 is activated but is not itself responsible for the activation.

Paternal care and the development of behavior in the southern grasshopper mouse, Onychomys torridus

Litters of southern grasshopper mice ( Onychomys torridus ) were reared by the male and female parents or by the female parent only. All mice were weaned at 25 ± 1 days of age and observed from 30 to 100 days of age in one of four different behavioral tests. These tests included: (a) open-field activity, (b) interspecific aggression toward Mus opponents of the same sex and same approximate age, (c) predatory behavior toward live house crickets, or (d) intraspecific aggression toward Onychomys opponents of the same sex and same approximate age. The presence of the male parent during the preweaning period resulted in a significant increase in the open-field activity of Onychomys females and a significant increase in the aggression of Onychomys males toward Mus opponents. In addition, there was a tendency for subjects reared by the male and female parents to be more efficient in attacking and killing cricket prey. There were no differences between subjects of the two rearing environments with respect to intraspecific aggression. These results suggest that the male parent had an important but selective influence on the behavioral development of Onychomys torridus males and females.