Loran L. Bieber

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.

A rapid spectrophotometric assay for carnitine palmitoyltransferase

Abstract The experimental conditions for a rapid and sensitive spectrophotometric assay for carnitine palmitoyltransferase (CPT) are described. The amount of reduced CoA liberated from palmitoyl CoA by CPT is quantitated using 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). Since corrections can be made for the amount of reduced CoA formed from palmitoyl CoA by other reactions, i.e., deacylations or transacylations, the assay can be used with impure CPT preparations such as liver mitochondria.

Scintillation counting of 32P without added scintillator in aqueous solutions and organic solvents and on dry chromatographic media

Abstract Cerenkov radiation produced by 32P makes possible a convenient and efficient method for determination of 32P in virtually any sample utilizing conventional scintillation counters. Organic solvents provide high absolute efficiencies—the highest efficiency measured, approximately 80%, was obtained with toluene. Approximately 50% absolute efficiency is obtained in aqueous solutions with very little variation when strong acids, bases, or salts are present. Paper and silica gel give similar efficiencies with no liquid present because Cerenkov radiation occurs in polyethylene and in glass. Samples are completely unaltered by the counting. Counting conditions are constant for all samples. The efficiency varies with volume, solution composition, and vial material. Efficiencies relative to water for a wide variety of aqueous solutions and organic solvents have been determined using a rapid, highly accurate method.

Differential increase of hepatic peroxisomal, mitochondrial and microsomal carnitine acyltransferases in clofibrate-fed rats.

Abstract Hepatic peroxisomes, mitochondria and microsomes from control and clofibrate-treated animals were separated by isopycnic sucrose gradient centrifugation and the carnitine acyltransferase system studied in each of these organelles. Clofibrate treatment produced a 13-fold increase in the total activity of carnitine acetyltransferase and a 5-fold increase in carnitine octanoyl- and palmitoyl-transferase activities. The specific activities of the transferases in all three subcellular locations increased, but to different extents. Peroxisomal and microsomal carnitine acetyltransferases doubled in specific activity; the mitochondrial enzyme increased 10-fold. Peroxisomal, mitochondrial and microsomal carnitine octanoyltransferases all increased 3-fold in specific activity. Carnitine palmitoyltransferase, which is found only in mitochondria, increased 3-fold in specific activity. These differential increases changed the per cent distribution of total carnitine acetyltransferase from 50 per cent in the mitochondria of control livers to 90 per cent in treated livers. Peroxisomes from clofibrate-treated livers had a consistently greater isopycnic density in sucrose gradients. Total catalase activity increased 2-fold upon treatment and a greater percentage of it was found in the paniculate fractions. The specific activity of peroxisomal catalase and urate oxidase remained the same as in controls. Carnitine acetyl- and octanoyltransferases are the first reported enzymes whose peroxisomal specific activity increases with clofibrate treatment. Preliminary results of treatment with another membrane-inducing drug, phenobarbital, indicated no change in peroxisomal density, catalase distribution and activity, and no effect on the specific activities of the peroxisomal, mitochondrial and microsomal carnitine acyltransferases.

Pivampicillin-promoted excretion of pivaloylcarnitine in humans

Pivampicillin treatment of seven children (five boys and two girls) for 7 days significantly reduced the amounts of total acid-soluble carnitine, free carnitine, and long-chain acylcarnitines and increased the amounts of acid-soluble acylcarnitine in plasma. The fasting plasma levels of 3-hydroxybutyrate at the end of treatment were 15% of the control value. The levels of free fatty acids were decreased, whereas triglyceride levels were unaffected, indicating impaired fat metabolism. Daily urinary excretion of total carnitine was four to five times higher than controls after the first day of treatment, although the amounts of free carnitine and acetylcarnitine were decreased. The urinary acylcarnitines were isolated and characterized by gas chromatography/electron impact mass spectrometry and fast-atom bombardment mass spectrometry. Pivaloylcarnitine was the predominant urinary acylcarnitine; it represented greater than 96% of the increased excretion of total carnitine and 75-80% of the total conjugated pivalic acid. The renal clearance of acylcarnitines was comparable to that of creatinine, indicating no reabsorption of pivaloylcarnitine. These data suggest a detoxification function of carnitine for pivalic acid in humans.

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.

Carnitine acyltransferases in rat liver peroxisomes

Abstract Carnitine acyltransferase activities, as well as acetyl-CoA, octanyl-CoA, and palmityl-CoA hydrolase activities, were assayed in mitochondrial, peroxisomal, and endoplasmic reticulum fractions after isopycnic density sucrose gradient fractionation of rat liver homogenates. Both the forward and reverse assays show that carnitine acetyltransferase and carnitine octanyltransferase are associated with peroxisomes, mitochondria, and endoplasmic reticulum, while carnitine palmityltransferase was detected in mitochondria. Palmityl-CoA and octanyl-CoA hydrolase activities were found in all but the leading edge of the peroxisome peak of the gradient. The palmityl-CoA hydrolase in peroxisomal fractions was due to lysosomal contamination since the activity coincided with the lysosomal marker, acid phosphatase. The substrate specificity for carnitine octanyltransferase activity was maximum with medium-chain-length derivatives (about 20 nmol/ min/mg protein) and decreased as the acyl length increased until very low activity (

Studies on the development of carnitine palmitoyltransferase and fatty acid oxidation in liver mitochondria of neonatal pigs.

Abstract Carnitine palmitoyltransferase (EC 2.3.1.23) levels were determined in liver mitochondria from 24-h-old fed and fasted, and 24-day-old piglets. After 24 h of age, carnitine palmitoyltransferase levels were more than double the level at birth and were about equal to the level at 24 days. Carnitine palmitoyltransferase activity increased faster in fed than in fasted animals. In contrast, the capacity of liver mitochondria from fed and fasted 24-h-old animals to oxidize added palmitoyl-coenzyme A in the presence of (−)-carnitine was only one-half that of 24-day-old animals. Evidence is presented that the distribution of carnitine palmitoyltransferase activity is about the same on each side of the long-chain acyl-coenzyme A barrier for liver mitochondria from newborn 1-day- and 5-day-old piglets. Calculations, based on the carnitine palmitoyltransferase levels and palmitoyl-coenzyme A oxidation rates, indicate that the dehydrogenase steps of fatty acid oxidation could provide sufficient electrons to maintain State -3 respiration rates of fatty acid oxidation in 1-day-old piglets. This indicates that carnitine palmitoyltransferase levels do not necessarily limit the rate of fatty acid oxidation in livers of 1-day-old piglets. Carnitine levels in several tissues from fed and fasted piglets were determined. After birth, liver carnitine levels double in 24 h in fed but not fasted animals. This increase is probably due to ingestion of sows milk which contains 0.13 mM (−)-carnitine.

A radioisotopic-exchange method for quantitation of short-chain (acid-soluble) acylcarnitines

A reliable and sensitive method for the quantitation of picomole amounts of acetylcarnitine, propionylcarnitine, aliphatic 4-carbon, and 5-carbon acylcarnitines has been developed. The procedure requires the measurement of the amounts of carnitine and acid-soluble carnitine and then the enzymatic exchange of 3H- or 14C-labeled L-carnitine into the acylcarnitine pool using commercial carnitine acetyltransferase, essentially free of acyl-CoA hydrolase activity. After isotopic equilibrium is obtained, the radioactive acylcarnitines are separated using either HPLC or thin-layer chromatography. Procedures for both are described. After separation, the amounts of radioactivity in the acylcarnitines are determined and the amount of individual acylcarnitines can be calculated from the specific activity of the initial total carnitine pool or from the ratio of dpm in the acylcarnitine fraction/dpm in free carnitine X (nanomoles L-carnitine) in the sample. The method has several advantages over current procedures, including rapidity, use of small sample sizes, simplicity, and reliability.