Albert A. Barber

Lipid peroxidation in rat tissue homogenates: Interaction of iron and ascorbic acid as the normal catalytic mechanism

Iron and ascorbic acid appear to be the normal catalytic components responsible for the lipid peroxidation reaction in aerobically incubated rat tissue homogenates. The amounts of each present in the catalytically-active fractions of rat liver, brain, testis, and kidney are appropriate to explain the lipid peroxidation reaction measured. Utilization of ascorbic acid as part of the normal catalytic mechanism is indicated by the following: The catalytic activity of the tissue soluble phase occurs only in the small molecule fraction eluted from Sephadex, and ascorbic acid occurs only in this fraction; the extent of catalysis by the small molecule fractions of the soluble phases from several tissues is proportional to their ascorbic acid content; and pH effect on lipid peroxidation is the same for both soluble-phase and ascorbic acid catalysis. Utilization of iron as part of the normal catalytic mechanism is indicated by EDTA inhibition studies and by measurements of pH effects. Previous studies have demonstrated the lack of catalytic activity by cations other than iron for the lipid peroxidation reaction in homogenates. Lipid peroxidation is inhibited at high tissue concentration and the inhibition is due to components occurring in the large molecule fraction of the soluble phase.

Age-related changes in membrane lipid content and enzyme activities

Abstract 1. 1. Microsomal and mitochondrial fractions were isolated from livers, kidneys and hearts of 6- and 24-month-old rats and the specific activities of several membrane-bound enzymes were determined. Differences were seen in the activities of the enzymes at the different ages. 2. 2. The phospholipid to protein ratios of the isolated fractions were determined. The phospholipid of the liver and kidney microsomal fractions was decreased in the old animals. No changes were seen in the ratio of membrane phospholipid to total lipid. 3. 3. Kinetic analysis was performed on two membrane-bound enzymes isolated from 6- and 24-month-old animals. Altered, perhaps inhibited, forms of the enzymes were indicated. 4. 4. It is concluded that enzyme changes observed to occur during aging are not directly correlated with in vivo phospholipid loss. The regulating influence of membrane conformation on the activity of bound enzymes, however, does appear to change.

Peroxidation of microsomal membrane protein—Lipid complexes

Nonenzymatic lipid peroxidation was studied using the TBA test on rat liver microsomal fractions, lipid micelles and structural protein-lipid micelle complexes. The kinetics, response to divalent cations, and iron-ascorbate catalysis were alike in the microsomal fraction and in the complex, but different in lipid micelles. The structural protein represented 41% of the total membrane protein, had a S20,obs of 3.5 and was hydrophobic. The binding of lipid micelles by structural protein proceeded in two steps, with an initial fast rate followed by a slower rate. The binding appeared to involve a hyrophobic association between lipid and protein as evidenced by insensitivity to pH, ionic strength and lack of preference for the individual classes of phospholipid micelles. Deoxycholate caused an increase in the initial peroxidation rate in microsomal fractions. Iron and ascorbate catalyzed lipid peroxidation in both the microsomal fraction and in the complex. Iron catalyzed lipid peroxidation but calcium, cobalt and copper inhibited the reaction in the SP-lipid micelle complex. Lipid peroxidation in microsomal suspensions, therefore, appears to be determined, in part, by the hydrophobic nature of the protein-lipid association found in membranes.

Lipid peroxidation in livers and kidneys from young and old rats.

Abstract Lipid peroxidation was measured in young (6 months) and old (24 months) rat tissue homogenates and microsomal fractions. Decreases in lipid peroxidation were noted in homogenates and microsomes from old animals. The decreases in liver and kidney appear due to reduced ascorbic acid content and the appearance of an inhibitor in the soluble phase. In addition old kidney microsomal fractions contain a membrane associated lipid soluble inhibitor. The significance of these inhibitors and of lipid-protein interactions in the regulation of lipid peroxidation are also discussed.

Inhibition of lipid peroxide formation by vertebrate blood serum.

Abstract Lipid peroxide formation in incubated rat brain homogenates was inhibited by all vertebrate blood sera tested. Rat and chicken sera contained both dialyzable and non-dialyzable inhibitors, whereas turtle, frog, and fish sera had only nondialyzable inhibitors. Chicken, turtle, and frog sera had two electrophoretically different inhibitors; rat and fish sera had only a single one. The inhibitor in rat was associated with an iron-binding protein believed to be siderophilin, and the mechanism of its action was chelation. The inhibitors of the other sera also seemed to be associated with the iron-binding proteins. More information on these proteins in the lower vertebrates is needed before the mechanism of action of these inhibitors can be established as conclusively as in the rat. The significance of these findings with respect to understanding the differences in radiation sensitivity of tissue antioxidant activity was discussed.

Oxygen binding characteristics of lobster hemocyanin and its subunits

Abstract 1. 1. Dissociation of lobster hemocyanin was broguht about by changes in pH. The effect of dissociation and reassociation of subunits on the oxygen equilibrium characteristics of hemocyanin was studied. Dissociation was determioned by ultracentrifugation and electrophoresis. Oxygen dissociation curves were obtained using a tonometer equipped with both a polarographic sensor and a 1 -cm quartz cuvette. 2. 2. The hemocyanin molecule remained aggregated between pH 4·5 and pH 8·5. Subuniting occured between both pH 3·0–4·5 and pH 8·5–10·5. The subunits sedimented with values of 8·5 S and 6·5 S , while the aggretated form of the molecule and an average value of 16·0 S . 3. 4. The reassociation of subunits was not complete, and although the autocatalytic nature of the ligand-binding reaction was not chagned in reassociated hemocyanin, the total amount of oxygen bound per molecule was slightly less. 4. 5. The numner of ligand-binding sites per molecule, as indicated by Hill equation analysis, is at least four.

Reconstitution of functional hemocyanin from apohemocyanin: The hepatopancreas as copper donor

Abstract 1. 1. Apohemocyanin was prepared from hemocyanin of the spiny lobster, Panulirus interruptus . 2. 2. The hepatopancreas of the lobster, which contains 75 per cent of the animals total copper, was centrifugally fractionated and the supernatant, which contains 90 per cent of the copper of the homogenate, was used as a copper donor for reconstitution of functional hemocyanin from apohemocyanin. 3. 3. Reconstitution occurred at pHs 5, 6 and 7. 4. 4. A low molecular weight fraction of the hepatopancreas supernatant fraction was the source of the copper. 5. 5. Copper replacement by the hepatopancreatic supernatant is non-enzymatic. 6. 6. Copper sulfate was also used to reconstitute hemocyanin. 7. 7. The reconstituted hemocyanin exhibited oxygen-binding characteristics identical to those of the native molecule.

COMPARATIVE HEMATOLOGY OF THE TURTLE, RABBIT AND RAT.

1. 1. The iron content of turtle blood was compared with that of the rabbit and rat. 2. 2. Serum iron and percent saturation of serum iron-binding proteins were lowest in the turtle. 3. 3. Differences in whole blood iron and hemoglobin concentrations were related to variations in total cell count, hematocrit and cell size. 4. 4. Turtle blood cells were found to contain a pool of non-hemoglobin iron not found in rabbit or rat. 5. 5. Only small amounts of iron were transferred from turtle iron-binding serum proteins to red cells during incubation in vitro.

A gel filtration method for studies on protein iron-binding

This communication reports the use of a gel filtration technique and radioactive iron for rapid determination of protein-iron binding in complex mixtures. Many methods have been used to investigate the characteristics of iron binding by mammalian serum. Calorimetric techniques have been used to measure the iron-binding capacities of whole seral. Electrophoretic2y3 and precipitation41s methods, using radioactive iron (6aFe), have confirmed the role of a /?-globulin, transferrin, as the primary iron-binding component of serum. Each of these techniques has certain limitations regarding specificity, binding interference, and rapidity of sample processing. Filtration through cross-linked dextran gels separates components of a complex mixture on the basis of molecular dimensions 6 8. The technique has been used for studies of the binding between proteins and small moleculesa*lo.

Kinetic analysis of the age related differences in glucose-6-phosphatase activity

Abstract Kinetic analysis was performed on glucose-6-phosphatase of 6 and 24 month old rat liver and kidney microsomal fractions. In untreated microsomal fractions of old liver the enzyme displayed decrease in V. In untreated microsomal fractions of old kidney and enzyme displayed decrease in V and increase in Km. Triton X-100 and NH4OH were used to disrupt the membranes and to activate the enzyme. The treatments lowered the Km of the enzyme. The activated enzymes of the young and old liver had similar Km but the old liver still displayed a lower V. The same results were found for the kidney. Kidney membranes were found to contain about 50% latent enzyme activity which was released by the treatments. The liver did not contain latent enzyme activity. The age related reduction in glucose-6-phosphatase activity appears to be due to reduction in the amount of enzyme present in the membranes. The membranes from old animals appear to be more labile to manipulation than the membranes from young animals.