Estelle J. McGroarty

Alpha hydroxy acid oxidation by peroxisomes

Abstract Peroxisomes from liver and kidney of rats and pigs were isolated by isopynic sucrose density gradient centrifugation. Alpha hydroxy acid oxidase was shown by electrophoresis to be a single liver peroxisomal protein which catalyzed the oxidation of glycolate and α-hydroxyisocaproate. With glycolate the Km was 2.4 × 10−4 m in rat liver and 1.3 × 10−3 m in pig liver, and V varied among peroxisomal preparations from 20–50 nmoles min−1 mg−1 protein. With α-hydroxyisocaproate the Km was 3.4 × 10−3 m , and V ranged from 20 to 30 nmoles min−1 mg−1 protein. The oxidase also catalyzed at a slower rate the oxidation of lactate, α-hydroxycaproate, and α-hydroxyvalerate. Peroxisomes from rat kidney contained one α-hydroxy acid oxidase which utilized α-hydroxyisocaproate and, to some extent, longer chain α-hydroxy acids, but did not oxidize glycolate or lactate. Peroxisomes from pig kidney contained two α-hydroxy acid oxidases, one for the short chain and the other for the long chain α-hydroxy acids, and both oxidized α-hydroxyisocaproate. The peroxisomal fraction, as isolated from rat liver homogenates, contained about 0.6% of the total lactate dehydrogenase, which may be as much as 1.5% if corrected for particle breakage. Less, but still substantial, lactate dehydrogenase was present in the rat kidney peroxisomal fraction. The lactate dehydrogenase isoenzyme pattern was the same for the peroxisomal and cytosol fractions in the rat, but not in the pig. The specific activity of lactate dehydrogenase from rat liver peroxisomes was 30% of that in the cytosol. No peak of lactate dehydrogenase was found in other fractions, such as the mitochondria. The results are inconclusive as to whether peroxisomes contain lactate dehydrogenase. However, as currently isolated on sucrose gradients, the peroxisomal fraction is so rich in lactate dehydrogenase activity, measured as pyruvate reductase, that it exceeds by 60- to 70-fold the α-hydroxy acid oxidase activity. Lactate dehydrogenase catalyzes the reduction of hydroxypyruvate and glyoxylate equally well. Mammalian peroxisomes do not contain a specific d -glycerate dehydrogenase or hydroxypyruvate reductase.

Glycerol phosphate dehydrogenase in mammalian peroxisomes

Abstract Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD + :α-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 70%, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviving intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min −1 mg −1 protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD + :α-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver peroxisomal and cytoplasmic NAD + :α-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands. Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1–2% of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K m values and migrated similarly to the anode at pH 6.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K m value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD + :α-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in animal peroxisomes.

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.

Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes

The opening and closing of the ompF porin from Escherichia coli JF 701 was investigated by reconstituting the purified protein into planar bilayer membranes. The electrical conductance changes across the membranes at constant potential were used to analyze the size and aggregate nature of the porin channel complexes and the relative number of opening and closing events. We found that, when measured at pH 5.5, the channel conductance diminished and the number of closing events increased when the voltage was greater than 100 mV. The results suggest that the number of smaller sized conductance channels increases above this potential. There was also an increase in the smaller subunits and in the closing events when the pH was lowered to 3.5, and these changes were further enhanced by increasing the voltage. We propose that both lowering the pH and elevating the potential across the membrane stabilize the porin in a conformation in which the subunits are less tightly associated and the subunits open in a non-cooperative manner. These same conditions also appear to stabilize the closed state of the pore.

A pH titration study on the ionic bridging within lipopolysaccharide aggregates

The packing of lipopolysaccharide aggregates from rough strains of Escherichia coli was examined at different pH values. Lipopolysaccharide head-group motion, measured with an electron spin resonance probe, was found to be dependent on pH, and indicated the existence of multiple ionizable groups. Lipopolysaccharide from a rough (Ra) and a heptose-less (Re) mutant were more rigid at pH 5 than at pH 10.5. In addition, head-group mobility of the magnesium salt of Ra lipopolysaccharide was substantially less than that of the sodium salt at pH 7.0, whereas at high pH (pH 12) the two salts were equally fluid. Changes in head-group packing were also reflected in pH-dependent changes in the phase transition measured with differential scanning calorimetry. The enthalpy of the transition, delta Ht, for the sodium salt of Re lipopolysaccharide was greatest at pH 7.5 and approached zero in both the acidic and the basic pH ranges. We propose that fixed charges in the core and lipid A regions significantly influence lipopolysaccharide head-group motion and the lipopolysaccharide aggregation state. Furthermore, ionic bridging among phosphate groups dramatically rigidifies head group interactions in the neutral to acidic pH ranges.

Acid PH decreases OMPF and OMPC channel size in vivo

To be effective against gram-negative organisms, beta-lactam antibiotics must be able to penetrate the outer membrane. For Escherichia coli, these compounds generally cross this barrier through non-specific channels in porins OmpF and OmpC. In vitro studies have shown that increased pH induces a switch in the structure of OmpF and OmpC from a small channel conformation to a set of larger-sized channel conformations. In this study, the permeability of two cephalosporins into cells producing either OmpC or OmpF was examined at various pHs. The results suggest that the pH-induced switch in channel size observed in vitro also occurs in vivo.

Correlation between temperature range of growth and structural transitions in membranes and lipids of Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild-type Escherichia coli grown at different temperatures were labelled with 1,6-diphenyl-1,3,5-hexatriene and analyzed using fluorescence polarization techniques. Lipids extracted from the membranes were similarly analyzed using fluorescence polarization. The thermotropic structural transition in outer membranes changed as a function of growth temperature. The structural transition in cytoplasmic membranes and lipids extracted from either cytoplasmic or outer membranes did not change with growth temperature. These data suggest that adaptive changes which occur in the outer membrane determine the temperature range of growth of E. coli. These changes apparently require alterations in outer membrane components other than phospholipids.

A cationic electron spin resonance probe used to analyze cation interactions with lipopolysaccharide

Abstract The partitioning of a cationic electron spin resonance probe, 4-(dodecyl dimethyl ammonium)-1-oxyl-2,2,6,6,-tetramethyl piperidine bromide, into lipopolysaccharide from Escherichia coli W1485 was shown to increase markedly above approximately 15°C, presumably reflecting a thermal transition. Partitioning was also highly dependent on probe and lipopolysaccharide concentrations, and Scatchard analysis of electrodialyzed lipopolysaccharide revealed a single non-interactive binding site for the probe. Several cations were able to displace probe bound to this site. At concentrations above 30 μM, Ca2+ and Mg2+ displaced probe bound to electrodialyzed lipopolysaccharide while various polyamines and other cations were less effective. Since this probe is very sensitive to the environment of the lipopolysaccharide, it should prove to be a valuable tool in analyzing lipopolysaccharide structure and interactions with other molecules.

Electron spin resonance probing of lipopolysaccharide domains in the outer membrane of Escherichia coli

Abstract (1) Scatchard analysis of the binding of the cationic electron spin resonance probe 4-(dodecyldimethylammonium)-1-oxyl-2,2,6,6-tetramethylpiperdine bromide to lipopolysaccharide and to phospholipid indicated that this probe has a 5 fold greater affinity for anionic lipopolysaccharide than for phospholipid. In the intact outer membrane this cationic probe likely associates with the lipopolysaccharide-containing outer monolayer. (2) The temperature dependence of the mobility of the cationic spin probe in the outer membrane indicates that a structural transition occurs at 9°C in the outer monolayer. (3) A similar 9°C transition was detected in the outer membrane using the spin probe 5-doxyl stearate. This anionic probe has been shown to preferentially partition into the phospholipid enriched domains of the outer membrane. (4) A porin-lipopolysaccharide-peptidoglycan complex probed with the cationic probe also was shown to undergo a thermally induced structural change at 9°C. (5) Purified lipopolysaccharide, however, was shown to have structural transitions at 20°C and 40°C. (6) It is proposed that a structural rearrangement of the intact outer membrane occurs at approx. 9°C in both the lipopolysaccharide and phospholipid domains of the outer membrane. Furthermore, this structural transition appears to be highly dependent on lipid-protein interactions. A second thermotropic transition that occurs in the outer membrane at approx. 40 to 42°C appears to result mainly from changes in the lipopolysaccharide domain structure.

Effects of aspirin, indomethacin, and sodium salicylate on human erythrocyte membranes as detected with electron spin resonance spectroscopy

Electron spin resonance spectroscopy of probed samples was used to determine the structural changes in human erythrocyte membranes prior to and at intervals following ingestion of either 10 grains acetylsalicylic acid, 10 grains sodium salicylate, or 50 mg indomethacin by both male and female subjects. Analysis of erythrocytes from female subjects indicated a time-dependent disordering of the membrane over the eight hour period following aspirin ingestion while the cells of male subjects showed a slight membrane ordering over the same time period. Erythrocytes drawn from females at the beginning of the menstrual cycle showed the greatest amount of membrane disordering at one hour following aspirin ingestion, but by eight hours, the membrane structure had returned to that of control. The time dependent disordering in membrane structure of cells from females in the middle of the menstrual cycle was biphasic. Ingestion of indomethacin induced only slight membrane changes in both male and female subjects over the times examined. Ingestion of sodium salicylate by either men or women did not induce significant changes in erythrocyte membrane order. Washed erythrocytes when mixed with salicylate, aspirin, or indomethacin were either identical to control cells or slightly more ordered. This study suggests that aspirin-induced alterations in membrane structure may depend upon steroid hormone levels.