N. E. Tolbert

A MODIFICATION OF THE LOWRY PROCEDURE TO SIMPLIFY PROTEIN DETERMINATION IN MEMBRANE AND LIPOPROTEIN SAMPLES

Abstract The original Lowry method of protein determination has been modified by the addition of sodium dodecyl sulfate in the alkali reagent and an increase in the amount of copper tartrate reagent. These alterations allowed the method to be used with membrane and lipoprotein preparations without prior solubilization or lipid extraction and with samples containing 200 m m sucrose or 2.5 m m EDTA.

[16] Protein determination in membrane and lipoprotein samples: Manual and automated procedures

Publisher Summary This chapter describes the protein determination in membrane and lipoprotein samples. A variety of methods have proved to be effective in estimating the protein content of water-soluble samples. The procedure used is based on this modified Lowry method. It is found that by adding sodium dodecyl sulfate to the alkali reagent, samples can be assayed directly without prior solubilization or delipidation. An increase in the copper tartrate concentration facilitates quantitation of protein in the presence of sucrose and EDTA. Color formation depends mainly on reduction of the Folin-Ciocalteu reagent by protein-bound copper and proceeds in two distinct steps. The wavelength used routinely in the modified Lowry procedure was chosen as a compromise between increased absorption of the final blue reduction product with longer wavelengths and the practical limitations of most spectrophotometers. The absorption peak of the blue chromophore extends through much of the visible spectrum in a broad plateau and reaches maximum at 750 nm. The effectiveness and rapidity of this modified Lowry procedure as compared to the original Lowry procedure for assaying complex biological systems are also discussed.

Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves

Abstract Isoenzymes of glucose-6-phosphate dehydrogenase and 6- P -gluconate dehydrogenase from a 70% ammonium sulfate precipitate of spinach leaf homogenate were separated by differential solubilization in a gradient of 70-0% ammonium sulfate and analyzed by disc gel electrophoresis. Isolated whole chloroplasts contained isoenzyme 1 of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase 1, whereas isoenzyme 2 of each was found in the soluble cytosol fraction. Both isoenzymes of each dehydrogenase were present in about equal amounts. Glucose-6-phosphate dehydrogenase isoenzymes 1 and 2 had pH optima of 9.2 and 9.0 and K m values of 400 and 330 μ m , respectively. Molecular weights for both isoenzyme of glucose-6-phosphate dehydrogenase were very similar at about 105,000 ± 10% as estimated by sedimentation velocity measurements. For 6-phosphogluconate dehydrogenase isoenzymes 1 and 2 the pH optima were 9.0 and 9.3, respectively, the K m values were 100 and 80 μ m , and the apparent molecular weights were also nearly identical at about 110,000 ± 10%. The data support the hypothesis that leaf cells have two oxidative pentose phosphate pathways, one in the chloroplast and the other in the cytosol.

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Abstract Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b 5 reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm −3 , a membrane fraction at 1.17 g × cm −3 containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient. Relative to other organelles the microbodies had a very low level of phospholipid (0.03–0.09 mg per mg protein). Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl serine were found in all organelle fractions. The percentage of each phospholipid in isolated microbodies from rat liver or spinach leaves was similar in the microbodies and microsomes. Glyoxysomes from germinating castor beans contained little phosphatidyl serine but rather an unidentified phospholipid. From spinach leaves the microsomes contained much phosphatidyl serine and the mitochondria had a significant amount of cardiolipin.

Glycolate dehydrogenase in green algae

Abstract Glycolate dehydrogenase was partially purified from Chlamydomonas by Triton X-100 extraction from the cells followed by ammonium sulfate fractionation. The enzyme had no oxidase activity. No glyoxylate formation occurred in the absence of the artificial electron acceptors, dichloroindophenol or phenazine methosulfate. The enzyme preferentially catalyzed the oxidization of glycolate at pH 8.0–8.7, and the Km was 2.2 × 10−4 m . Glycolate dehydrogenase also catalyzes the oxidation of d -lactate with a Km of 1.5 × 10−3 m . The enzyme was sensitive to sulfhydryl inhibitors. No cofactors have been identified. A limited survey was run for the distribution of the two enzymes which catalyze the oxidation of glycolate. Glycolate oxidase couples to oxygen, is cyanide-insensitive, and oxidizes l -lactate. In contrast, glycolate dehydrogenase is cyanide-sensitive and oxidizes d -lactate. Glycolate dehydrogenase activity was found in the green algae, Chlamydomonas, Chlorella, Scenedesmus, Euglena, and Acetabularia, and no glycolate oxidase was detected in these algae. Higher plants had glycolate oxidase activity but no glycolate dehydrogenase. It is suggested that the enzyme for oxidizing glycolate to glyoxylate is a dehydrogenase in algae and an oxidase in the peroxisomes of higher plants.

Formotion of serine and glyceric acid by the glycolate pathway

Abstract Distribution of C 14 in the C 3 compounds, phosphoglycerate, glycerate, alanine, and serine from five different species of plants was determined after 20 sec. of photosynthesis with C 14 O 2 . In each experiment phosphoglycerate, glycerate, and alanine were predominately carboxyl labeled, but serine was uniformly labeled. Thus, serine biosynthesis must proceed by a different route than that for the other C 3 compounds. Since glycerate was labeled less in the carboxyl carbon than phosphoglycerate, part of the glycerate may have been synthesized from a precursor other than carboxyl-labeled phosphoglycerate. When glycolate-2-C 14 was fed to the same plants, serine-2,3-C 14 and glycerate-2,3-C 14 were major products. When serine-3-C 14 was fed, glycerate-3-C 14 was formed. The C 14 labeling was consistent with a glycolate pathway which started with phosphoglycolate and glycolate and led to the synthesis of glycine, then serine, and finally glycerate. This sequence was substantiated by studies on the time and rate of labeling of these products during glycolate-2-C 14 metabolism. The significance of the glycolate pathway was indicated by the high percentage of fixed C 14 which was found in the products of the pathway after brief periods of photosynthesis with C 14 O 2 . A diurnal variation was observed for the ratio of C 14 photosynthetically incorporated into serine compared to glycine.

Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules.

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.

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.

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Abstract Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P2) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P2, is also present. Both compounds simulate the substrate inhibition of ribulose-P2 carboxylase/oxygenase previously reported for ribulose-P2. Freshly prepared ribulose-P2 had little inhibitory activity. The instability of ribulose-P2 may be one reason for a high level of ribulose-P2 carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P2. Because the KD of the enzyme/substrate complex is ≤1 μM, all ribulose-P2 generated in situ may be stored as this complex to prevent decomposition.

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Abstract Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase. Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O -(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes. Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.