Sharron L. Nance

Initial electron transfer in photosynthetic reaction centers of Rhodobacter capsulatus mutants

Abstract The stimulated emission decay time constants were measured for a series of Rhodobacter capsulatus reaction centers with site-specific mutations at the symmetry related locations M208 and L181. We report the first mutant (Phe L181 → Tyr) that exhibits an initial electron transfer rate faster than the native organism at 295 K, and determine that the tyrosine at position M208 cannot be fully responsible for the unidirectionality of electron transfer.

Liposome-Encapsulated Actinomycin D: Potential in Cancer Chemotherapy

Summary Actinomycin D, when encapsulated within liposomes, is less toxic to mice than the nonencapsulated form. A single dose (0.75 mg/kg) or multiple doses (1 × 0.50, 4 × 0.25 mg/kg) significantly increased the mean survival time of mice inoculated with Ehrlich ascites tumor cells. Liposomes containing actinomycin D were found in tumor cells and cell degeneration and death were subsequently observed.

Spectrophotometric Assay of Catalase with Perborate as Substrate

Summary A spectrophotometric assay for catalase has been developed in which sodium perborate, rather than hydrogen peroxide, is used as the substrate. The method is convenient, rapid, and readily adapted to the measurement of catalase in subcellular fractions.

Second-site mutation at M43 (Asn→Asp) compensates for the loss of two acidic residues in the QB site of the reaction center.

Two acidic residues, L212Glu and L213Asp, in the QB binding sites of the photosynthetic reaction centers of Rhodobacter capsulatus and Rhodobacter sphaeroides are thought to play central roles in the transfer of protons to the quinone anion(s) generated by photoinduced electron transfer. We constructed the site-specific double mutant L212Ala-L213Ala in R. capsulatus, that is incapable of growth under photosynthetic conditions. A photocompetent derivative of that strain has been isolated that carries the original L212Ala-L213Ala double mutation and a second-site suppressor mutation at residue M43 (Asn→Asp), outside of the QB binding site, that is solely responsible for restoring the photosynthetic phenotype. The Asp,Asn combination of residues at the L213 and M43 positions is conserved in the five species of photosynthetic bacteria whose reaction center sequences are known. In R. capsulatus and R. sphaeroides, the pair is L213Asp-M43Asn. But, the reaction centers of Rhodopseudomonas viridis, Rhodospirillum rubrum and Chloroflexus aurantiacus reverse the combination to L213Asn-M43Asp. In this respect, the QB site of the suppressor strain resembles that of the latter three species in that it couples an uncharged residue at L213 with an acidic residue at M43. These reaction centers, in which L213 is an amide, must employ an alternative proton transfer pathway. The observation that the M43Asn→Asp mutation in R. capsulatus compensates for the loss of both acidic residues at L212 and L213 suggests that M43Asp is involved in a new proton transfer route in this species that resembles the one normally used in reaction centers of Rps. virddis, Rsp. rubrum and C. aurantiacus.

Isotope and solvent effects of deuterium on aconitase.

Abstract The activity of aconitase has been compared in H 2 O and in 99% D 2 O, with deuteriocitrate and deuterioisocitrate as well as the protiated substrates, citrate, isocitrate, and aconitate. The rates of all six interconversions were markedly inhibited by D 2 O; the conversion of aconitate to citrate was affected much more than the conversion of aconitate to isocitrate. The values obtained for Michaelis constants and maximum velocities satisfactorily predict equilibrium conditions in H 2 O and D 2 O. Negligible isotope effects were observed with deuteriocitrate or deuterioisocitrate; this observation is consistent with the hypothesis that the first (and rate-limiting) step in the dehydration of these compounds is the rupture of the CO rather than the CH bond.

Studies on rat liver ribonucleases: II. Zonal centrifugation of acid ribonuclease; implications for the heterogeneity of lysosomes

Abstract Results obtained by zonal gradient centrifugations demonstrated that rat liver lysosomes are heterogeneous in terms of their enzyme contents. It is suggested that acid phosphatase and cathepsin C on one hand, and acid ribonuclease and cathepsin D on the other, belong to two different classes of “lysosomes”.

Some metabolic characteristics of brown fat, with particular reference to the mitochondria☆

Abstract 1. 1. Mitochondria from the axillary brown adipose tissue of euthermic hibernating and newly-aroused ground squirrels (Citellus tridecemlineatus) have been prepared and assayed for a number of enzyme systems. 2. 2. The sedimentation behavior of brown fat mitochondria is considerably different from that of liver mitochondria, although the equilibrium density, measured by isopycnic centrifugation, is the same for both. 3. 3. Although the respiratory capacity of brown fat mitochondria is very high, they lack the capacity (a) to esterify inorganic phosphate, (b) to accumulate calcium phosphate or (c) to undergo reversibility of swelling induced by thyroxin or glutathione. 4. 4. The intracellular distribution of various enzymes in brown fat is qualitatively similar to that seen in other tissues; although the mitochondria-linked enzymes are present to a significantly greater extent in brown fat than in liver, the concentrations of non-mitochondrial enzymes such as acid phosphatase, catalase and glucose-6-phosphatase are much lower. 5. 5. In general, enzymatic activities of brown fat were somewhat higher in the aroused, and lower in the hibernating ground squirrels, in comparison with the euthermic animals.

OXIDATIVE PHOSPHORYLATION IN LIVER MITOCHONDRIA FROM X-IRRADIATED RATS

The extent of the effects of ionizing radiation on structure and function of liver mitochondria has been the subject of a large number of communications. For example, Noyes and Smith (1) reported that within 15 minutes after exposure of male rats to 1000 R of Co60 y-radiation there was a marked depression of mitochondrial numbers, with a concomitant increase in unit mass per mitochondrion. This effect persisted about 90 minutes and then diminished, and by 6 hours after irradiation both the number and the size of mitochondria were normal. Hall et al. (2) described a similar effect in male rats receiving 840 R of X-radiation; although the decrease in number was not so marked, the recovery was more protracted, and the increase in total nitrogen per mitochondrion observed by Noyes and Smith was not noted in these studies. Hall et al. (2) also observed significant changes in oxidative phosphorylation during the first 12 hours after exposure, the P/O ratios (micromoles of orthophosphate esterified per microatom of oxygen consumed) falling to 70 to 75 % of the control values, then returning to normal at 24 hours. This decrease appeared to represent a true uncoupling, oxygen consumption increasing and phosphate esterification decreasing. Inhibition of oxidative phosphorylation in rat liver mitochondria was observed 24 hours after irradiation by Ryser et al. (3) and by Yost and his co-workers (4, 5); the former group observed no effect 2 hours after exposure, whereas the latter made no mention of experiments at earlier times. Nitz-Litzow and Biihrer also reported a 30% decrease in P/O ratios 12 hours after 800 R (6). Profound but transitory changes in phosphorylation, respiration, and swelling characteristics of mouse liver mitochondria 6 hours after total-body irradiation with 500 R have been described by Urakami (7). By the use of density-gradient centrifugation of rat liver homogenates, Thomson and Rahman (8) were unable to detect any changes in size distribution of mito-

The unique distribution of ornithine aminotransferase in rat liver mitochondria.

Abstract Mitochondria were isolated from normal rat and mouse liver and kidney, hepatocytes, ascites tumor cells, and livers of rats treated in various ways known to affect the size of liver mitochondria. These mitochondria were then distributed by particle size through a linear sucrose-density gradient in a zonal centrifuge, and the distribution of ornithine aminotransferase activity was compared to that of malate dehydrogenase activity. While the activities of these enzymes are congruent in mitochondria prepared from several tissues other than liver, the midpoints of their distributions are consistently different in mitochondria prepared from whole liver and from hepatocytes. The changes in the separation of midpoints in animals treated in various ways (subtotal hepatectomy, corticosteroid administration, and diet modification) are consistent with the histologic effects of these treatments on liver mitochondria, and suggest that ornithine aminotransferase activity may prove a useful marker for a unique population of mitochondria perhaps localized in cells near the central vein.

STUDY OF REACTION CENTER FUNCTION BY ANALYSIS OF THE EFFECTS OF SITE-SPECIFIC AND COMPENSATORY MUTATIONS

The protein portion of the reaction center (RC) complex is composed of three subunits: the intermembrane L and M chains, and the cytoplasmic H polypeptide. The cofactors are within the transmembrane region; they are related by approximate twofold symmetry as are the homologous L and M chains [1–4]. The cofactors consist of a bacteriochlorophyll dimer that is the primary electron donor, two bacteriochlorophyll monomers, two bacteriopheophytins, a non-heme iron atom, and two quinones which serve as the final electron acceptors.