Production Of Apoprotein Research Articles

Controlling the delicate balance of tetrapyrrole biosynthesis

Tetrapyrroles are a family of compounds that contain four pyrrole rings. They are involved in many fundamental biological processes such as photoreception, electron transport, gas transport and also as cofactors for enzymatic reactions. As regulators of protein activity, tetrapyrroles mediate cellular response to light, oxygen and nutrient levels in the surrounding environment. Biosynthesis of haem tetrapyrroles shares, conserved pathways and enzymes among all three domains of life. This is contrasted by chlorophyll biosynthesis that is only present in eubacteria and chloroplasts, or cobalamin biosynthesis that is only present in eubacteria and archaea. This implicates haem as the most ancient, and chlorophyll as the most recent, of the common tetrapyrroles that are currently synthesized by existing organisms. Haem and chlorophyll are both toxic when synthesized in excess over apo-proteins that bind these tetrapyrroles. Accordingly, the synthesis of these tetrapyrroles has to be tightly regulated and coordinated with apo-protein production. The mechanism of regulating haem and chlorophyll synthesis has been studied intensively in Rhodobacter species and will be discussed.

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.

Surfactant Apoprotein D in Preterm Neonates with Acute Respiratory Distress Syndrome

Objective: to study the production of surfactant apoprotein D in preterm neonates with acute respiratory distress syndrome (ARDS) during artificial ventilation (AV). Subjects and methods. The paper presents the results of studying the production of surfactant protein D (SP-D) in various biological fluids in 44 preterm neonates. Two groups of newborn infants were identified according to the clinical manifestations of ARDS. The study group comprised 25 infants with the severe course of the disease, in this connection the preventive administration of the exogenous surfactant Curosurf and AV were made in all the neonates at birth. The control group included 19 preterm babies without signs of ARDS. Results. The study has demonstrated that in parturients and preterm neonatal infants, surfactant apoprotein D is detectable in various biological fluids: amniotic fluid, the gastric aspirate obtained just after birth, residual umbilical cord blood, serum following 8 hours of birth, and bronchoalveolar fluid. Despite the low gestational age of the neonates, the lung surfactant system is able to produce SP-D, as evidenced by its high content in the amniotic fluid and residual umbilical cord blood of preterm neonates. The production of apoprotein D in preterm neonates considerably reduces in the next few hours after birth. Conclusion. The findings suggest that fetal tissues generate SP-D, which improves pulmonary gas exchange in preterm neonates in the first hours after birth and that alveolar-capillary membrane dysfunctions are transient in the neonates on AV. Key words: preterm neonates, acute respiratory distress syndrome, surfactant, surfactant apoprotein D.

Effects of alcohol and cholesterol feeding on lipoprotein metabolism and cholesterol absorption in rabbits.

Alcohol fed to rabbits in a liquid formula at 30% of calories increased plasma cholesterol by 36% in the absence of dietary cholesterol and by 40% in the presence of a 0.5% cholesterol diet. The increase was caused almost entirely by VLDL, IDL, and LDL. Cholesterol feeding decreased the fractional catabolic rate for VLDL and LDL apoprotein by 80% and 57%, respectively, and increased the production rate of VLDL and LDL apoprotein by 75% and 15%, respectively. Alcohol feeding had no effect on VLDL apoprotein production but increased LDL production rate by 55%. The efficiency of intestinal cholesterol absorption was increased by alcohol. In the presence of dietary cholesterol, percent cholesterol absorption rose from 34.4+/-2.6% to 44.9+/-2.5% and in the absence of dietary cholesterol, from 84.3+/-1.4% to 88.9+/-1.0%. Increased cholesterol absorption and increased LDL production rate may be important mechanisms for exacerbation by alcohol of hypercholesterolemia in the cholesterol-fed rabbit model.

Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli

P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5′-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by K m were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity.

Dietary fatty acids and dietary cholesterol differ in their effect on the in vivo regulation of apolipoprotein A-I and A-II gene expression in inbred strains of mice

Dietary cholesterol and dietary saturated fatty acids affected the plasma concentrations of various HDL components and the hepatic and intestinal expression of the apolipoprotein (apo) A-I gene and the hepatic expression of the A-II gene differently in three inbred strains of female mice. Thus, the HC diet (0.5% cholesterol, no added fatty acids) decreased HDL-cholesterol in C57BL and SWR strains but not in the C3H strain; plasma apo A-I and apo A-II concentrations decreased in all three strains. HDL-C/apo A-I and apo A-I/apo A-II mass ratios increased, suggesting that the HC diet altered both the concentrations and the compositions of HDL particles. In contrast, the HF diet (20% hydrogenated coconut oil, no added cholesterol) increased HDL cholesterol and apo A-I concentrations. The combination diet (HF/C, 20% coconut oil plus 0.5% cholesterol) increased HDL cholesterol and decreased triacylglycerols. Apo A-I concentrations were unaltered except for a significant increase in SWR mice. Apo A-II concentrations decreased in all strains. To examine molecular events that could lead to the changes in plasma apo A-I and apo A-II, we measured transcription rates in hepatic nuclei and steady state mRNA concentrations in liver and intestine and apo A-I synthetic rates in liver. Dietary cholesterol and fatty acids produced differing effects at transcriptional as well as post-transcriptional loci and the changes differed according to mouse strain. The most pronounced strain−related differences for both apo A-I and apo A-II occurred at post-transcriptional loci of apoprotein production. These could represent altered rates of translation in, or secretion from liver and/or intestine, or altered rates of clearance from plasma. In conclusion, the regulation of apo A-I and apo A-II gene expression by diet occurs at several steps of their production and perhaps also in catabolic pathways. This study identifies potential loci of regulation and forms the basis for future studies investigating specific genetic and molecular regulatory mechanisms.

Serum Cortisol Concentrations in Children with Protein-Energy Malnutrition

ABSTRACT We measured basal Cortisol levels in 15 children with kwashiorkor, 15 children with marasmic kwashiorkor, and 21 children with marasmus, before and after nutritional rehabilitation, as wel...

Differential effects of cyclosporin on hepatic and renal heme, cytochrome P-450 and drug metabolism: Possible role in nephrotoxicity of the drug

Treatment of rats with 25 or 50 mg/kg cyclosporin A for 6 days elicited vastly different responses in hepatic and renal heme and drug metabolism activities. In the liver, cytochrome P-450 concentration was decreased significantly (to 70–75 % of the control). This was accompanied by a marked reduction in benzo[ a]pyrene hydroxylase activity (to 20–28% of the control). Aniline hydroxylation was also decreased, but to a lesser extent (to 77% of the control). In contrast, in the kidney cytochrome P-450 concentration was significantly increased to (145–170% of the control), along with a modest decreasc in benzo[ a]pyrene hydroxylation activity. In this organ, the concentration of porphyrins was severely decreased (to 30% of the control). Also, the activities of δ-aminolevulinate (ALA) synthetase and ALA dehydratase, as well as that of heme oxygenase, were inhibited. It is suggested that in the kidney the inhibition of degradation, rather than an enhanced rate of synthesis of the heme molecule, contributes to the observed increase in cytochrome P-450 concentration. In the liver, the decrease in the cytochrome concentration could not be explained in terms of an alteration in the rate of heme biosynthesis or degradation. Therefore, the observed decrease in cytochrome P-450 concentration could reflect the direct inactivation of the hemoprotein or regulation of apoprotein production by cyclosporin and/or its metabolites(s). The possible relevance of the observations to cyclosporin nephrotoxicity is discussed.

Metabolic heterogeneity of low density lipoprotein-apoB production in familial hypercholesterolemia: an analytical model solution of tracer data.

Six subjects with heterozygous familial hypercholesterolemia (FH) (three males, three females) formed the basis for an investigation of the pathways of production of apoprotein B within plasma low-density-lipoprotein (LDL-apoB). Following the intravenous injection of [75Se]selenomethionine as an amino acid tracer, incorporation of the radioactive isotope into the putative precursor of LDL, intermediate density lipoprotein (IDL-apoB), was examined over a 9-day period. The resulting tracer data provided the precursor profile for both the IDL catabolic conversion of LDL as well as for direct synthesis of LDL from amino acids. The fractional conversion rates, (beta) for IDL and (alpha) for amino acids to LDL-apoB, were determined utilizing the two-compartment model for LDL involving both the intravascular and the extravascular pools of apoB. This LDL model was resolved analytically and the parameters alpha and beta were determined so as to give the least squares fit to the LDL tracer data. In this solution, the fractional conversion rates of IDL and of amino acids to LDL-apoB were resolved with a mean fractional residual of 20 +/- 6%, which was randomly distributed within the LDL-apoB data throughout the 216 hours of the studies. The steady-state determination of the pathways of LDL-apoB synthesis, based upon this tracer analysis, indicate that IDL accounts for 46%, and amino acids account for 54% of total apoB production within plasma LDL in heterozygous FH. In contrast, in normal subjects, IDL accounts for 86% of total LDL-apoB production. Since the IDL catabolic pathway represents the predominant if not exclusive source of LDL in normal man, this demonstration of a major non-IDL source of LDL in FH broadens the understanding of the pathophysiology of human familial hypercholesterolemia.

Metabolism of apolipoproteins CII, CIII 1, CIII 2 and VLDL-B in human subjects consuming high carbohydrate diets

Simultaneous kinetics of apoproteins CII, CIII 1 and CIII 2 and VLDL-B were determined in six normal subjects initially while consuming a control diet and subsequently while consuming a high carbohydrate diet. Mean triglyceride levels in plasma and VLDL were significantly increased, from 110 to 173 mg/dl and 40 to 114 mg/dl respectively, by the high carbohydrate diet. Specific radioactivity of each C peptide was determined, following injection of 125I-VLDL, in both VLDL and HDL from plasma samples taken over 48 hr using a technique involving analytical isoelectric focusing. Specific activity vs time curves for VLDL and HDL were almost superimposable on either diet, suggesting that CII, CIII 1 and CIII 2 were catabolized as a group. The high carbohydrate diet resulted in a significant increase in the mass of each C peptide which was brought about by an increase in production since no change in fractional removal rate (FRR) was observed. The pool size of VLDL apo B was also significantly increased and was a function of both increased production as well as decreased FRR. The proportion of CIII 2 mass relative to those of CII and CIII 1 was lower in both VLDL and HDL with the high carbohydrate diet. These observations indicate that the metabolism of the B and C apoproteins are independent of each other and do not respond in the same manner to changes in VLDL triglyceride and that increased triglyceride synthesis and secretion stimulates C apoprotein production.

Different kinetic fates of apolipoproteins A-I and A-II from lymph chylomicra of nonhuman primates. Effect of saturated versus polyunsaturated dietary fat.

Monkeys fed polyunsaturated fat had significantly lower plasma cholesterol (186 +/- 18 vs. 276 +/- 31 mg/dl) and high density lipoprotein (HDL) mass concentrations (466 +/- 28 vs. 518 +/- 34 mg/dl) than did animals fed saturated fat. Plasma apoA-I concentrations also were significantly lower and apoA-II levels were generally, though not significantly, lower in the group fed polyunsaturated fat. In vivo reinjection studies, using thoracic duct lymph chylomicra labeled with (131)I and HDL labeled with (125)I, were done in order to study the mechanism of plasma HDL-lowering by polyunsaturated dietary fat. The peak specific activity (SA) of HDL apoA-I derived from (131)I-labeled chylomicra occurred at 3 hr after injection (172 +/- 11% of 1 min S.A.) and then an exponential decay occurred indicative of a precursor-product relationship between chylomicron apoA-I and HDL apoA-I. In contrast, HDL apoA-II derived from (131)I-labeled chylomicra had no early S.A. increase and began to die away immediately after injection. Labeled apoA-I from chylomicron and HDL origin had similar plasma fractional catabolic rates (FCR = 0.34-0.38 vs. 0.32-0.38 d(-1), respectively); apoA-II from chylomicron or HDL origin also had similar FCR (0.46-0.51 vs. 0.42-0.51 d(-1), respectively), which were significantly shorter than those for HDL apoA-I. There was a consistent trend toward a higher FCR for HDL apoA-I or A-II of polyunsaturated fat-fed recipients. Chylomicron apoA-I/triglyceride and apoA-II/triglyceride mass ratios were lower in polyunsaturated fat-fed animals (A-I/TG = 1.56 x 10(-3); A-II/TG = 1.47 x 10(-3)) vs. saturated fat-fed animals (A-I/TG = 2.58 x 10(-3); A-II/TG = 2.77 x 10(-3)). It was concluded that: (1) dietary polyunsaturated fat significantly lowered plasma cholesterol, HDL, and apoA-I concentrations relative to saturated fat; (2) the HDL-lowering effect of the dietary polyunsaturated fat may be due to the combined effects of decreased apoprotein production by the intestine and increased HDL catabolism; and (3) in the blood, chylomicron apoA-I and A-II differ in their metabolic fates.-Parks, J. S., and L. L. Rudel. Different kinetic fates of apolipoproteins A-I and A-II from lymph chylomicra of nonhuman primates. Effect of saturated versus polyunsaturated dietary fat.

Mechanisms of intestinal fat absorption.

Even though most of the major controversies regarding intestinal fat absorption have been resolved over the past three decades, our concepts of fat digestion and absorption continue to be modified, and new concepts have emerged. The purpose of this review is to summarize advances in our understanding of fat digestion and absorption since the topic was last reviewed by Johnston in 1968. The discussion will emphasize 1) the role of colipase and its interactions with lipase, bile salt micelle, and triglyceride substrate; 2) the importance of the unstirred water layer in fat absorption; 3) micellar formation and dissociation; 4) the role of fatty acid binding protein; 5) factors influencing the reesterification mechanisms in the intestine; and 6) intestinal contribution to lipoprotein and apoprotein production. The importance of these new concepts and the remaining gaps in our understanding of these complex digestive and absorptive processes are discussed.

Turnover of apoproteins A-I and A-II of high density lipoprotein and the relationship to other lipoproteins in normal and hyperlipidemic individuals

The kinetics of the major apoproteins of high density lipoproteins (HDL), A-I(apoA-I) and A-II(apoA-II), were studied from the specific activity-time curves of these apoproteins, after reinjection of radioiodine-labeled HDL. In all 20 subjects, HDL apoprotein kinetics conformed to a two-pool model. The total fractional removal rates for the two apoproteins were similar, although the irreversible fractional removal rate appeared to be slightly greater for apoA-I. The mean transport for A-I and A-II was 12.2 mg/kg/day and 5.0 mg/kg/day, respectively. The mass of the apoprotein pools was strongly correlated with apoprotein production rate and also, to a lesser degree and inversely, with the irreversible fractional catabolic rate. Transport was directly correlated with body weight. Higher fractional catabolic rates, including the transfer rates between the two pools, were observed in five hypertriglyceridemic subjects: in contrast, five subjects with familial hypercholesterolemia tended to show lower fractional catabolic rates. These findings were supported by (1) a strongly positive correlation between the transport rates of HDL A-I and of very low density lipoprotein (VLDL) apoB, determined simultaneously in 10 subjects: and (2) a significant inverse correlation between the irreversible fractional removal rate of HDL A-I and the concentration of low density lipoprotein (LDL) apoB, measured in 15 subjects. These observations underline the metabolic interrelationships of the major lipoprotein classes. Two subjects with familial hyperalphalipo-proteinemia showed enlarged pool sizes, but normal transport, with irreversible fractional removal rates that were in the lower range for the group of 20 subjects.

Plastocyanin : Possible Significance of Quaternary Structure

Some properties of the blue copper protein plastocyanin from the green alga Scenedesmus have been investigated and compared with that from spinach, including amino acid composition, isoelectric point and copper content. The protein from Scenedesmus contains two, that from spinach four copper atoms per molecular weight of 40000. A combination of sodium dodecylsulfate/polyacrylamide gel electrophoresis and quantification of sulfhydryl groups indicates a strong preference for a species composed of 4 polypeptide chains of identical amino acid composition representing the enzymically active entity. Due to various treatments the subunits of both plastocyanins are detected as either monomer species alone or as monomer and dimer in a molar ratio of 2 : 1 on sodium dodecylsulfate gels. The four -SH groups per molecule are of different reactivity: two -SH groups can be detected after destruction of the chromophore; two more (forming an S-S bridge in the dimer) become evident after appropriate reduction. A KCN treatment for production of apoprotein is reported and the use of electrodialysis to improve incomplete apoprotein formation. These studies lend support to the proposal of a quaternary structure. Apoproteins were subjected to dodecylsulfate gel analysis, which proved to be an effective means of estimating both the extent of apoprotein formation and its reconstitution to the haloprotein.

Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum.

The role of Mo in the activity and synthesis of the nitrogenase components of Clostridium pasteurianum has been studied by observing the competition of Mo with its structural analogue W. Clostridial cells when fixing N2 appeared strictly dependent upon the available Mo, showing maximal N2-fixing activity at molybdate concentrations in the media of 10 muM. Cells grown in media with 3 times 10(-6) muM Mo, although showing good growth, had only 15% as much N2-fixing activity. In the presence of W the synthesis of both nitrogenase components, molybdoferredoxin and azoferredoxin, was affected. Attempts to produce nitrogenase in W-grown cells by addition of high molybdenum to the media in the presence of inhibitors of protein synthesis showed that Mo incorporation into a possible inactive preformed apoenzyme did not occur. Unlike other molybdoenzyme-containing cells, in which W either is incorporated in place of Mo to yield inactive protein or initiates the production of apoprotein, C. pasteurianum forms neither a tungsten substituted molybdoferredoxin nor an apoprotein. It is concluded that in C. pasteurianum molybdenum is an essential requirement for both the biosynthesis and activity of its nitrogenase.