Gradient gel electrophoresis-immunoblot analysis (GGEI): a sensitive method for apolipoprotein profile determinations

A method is described which will determine the distribution of individual apolipoproteins within the HDL subclasses. This method requires 1-2 microliters of plasma per determination and involves six steps: 1) electrophoresis of samples on non-denaturing 2-30% concave acrylamide gradient gels; 2) electrophoretic transfer of the lipoproteins to charge-modified nylon membranes; 3) fixation of the transferred lipoproteins with glutaraldehyde; 4) immunolocalization of the apolipoproteins with iodinated monospecific antibodies; 5) autoradiography followed by densitometry; and 6) reduction of the data to provide a plot of percent distribution versus particle size. When this method was applied to the analysis of rat apolipoproteins, differences were noted in the distribution of apoA-I, apoA-IV, and apoE. The majority of apoA-I was localized to HDL particles between 9 and 12 nm in diameter, with a median diameter of 10.0 nm, while apoE resided on substantially larger particles with a median diameter of 12.5 nm. ApoA-IV could be localized to three distinct areas: an HDL particle with a median diameter approximately 0.4 nm larger than apoA-I HDL, a particle smaller than albumin (lipoprotein-free apoA-IV), and a particle of 7.6 nm that does not appear to contain apoA-I or apoE.     

ApoA-IV metabolism in the rat: role of lipoprotein lipase and apolipoprotein transfer

Factors influencing the association of apoA-IV with high density lipoproteins (HDL) were investigated by employing a crossed immunoelectrophoresis assay to estimate the distribution of rat plasma apoA-IV between the lipoprotein-free and HDL fractions. Incubation of rat plasma at 37 degrees C resulted in the complete transfer of lipoprotein-free apoA-IV to HDL within 45 min. When plasma obtained from fat-fed rats was incubated at 37 degrees C in the presence of postheparin plasma as a source of lipolytic activity, there was a complete transfer of HDL apoA-IV to the lipoprotein-free fraction within 30 min. With extended incubation (120 min), lipoprotein-free apoA-IV began to transfer back to HDL. Similar patterns of apoA-IV redistribution were seen when plasma from fat-fed rats was incubated with postheparin heart perfusate or was perfused through a beating heart. Incubations conducted with plasma obtained from fasted rats showed similar but markedly attenuated apoA-IV responses. Similar observations were found in vivo following intravenous heparin administration. To determine whether the transfer of apolipoproteins from triglyceride-rich lipoproteins to HDL was partially responsible for the lipolysis-induced redistribution of apoA-IV, purified apoA-I, apoE, and C apolipoproteins were added to plasma from fasted rats. When added to plasma, all of the apolipoproteins tested displaced apoA-IV from HDL in a dose-dependent manner. Conversely, apolipoproteins were removed from HDL by adding Intralipid to plasma from fasted rats. With increasing concentrations of Intralipid, there was a progressive loss of HDL apoC-III and a progressive increase in HDL apoA-IV. Intravenous injection of a bolus of Intralipid to fasted rats resulted in a transient decrease of HDL apoC-III and concomitant increase in HDL apoA-IV. From these studies, we conclude that the binding of apoA-IV to HDL is favored under conditions that result in a relative deficit of HDL surface components, such as following cholesterol esterification by LCAT or transfer of apolipoproteins to nascent triglyceride-rich lipoproteins.     

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Intracellular microinjection of alkaline buffers reversibly inhibits the initial phase of hormone action in meiosis reinitiation of starfish oocytes

Intracellular microinjection of alkaline Hepes-KOH buffers, which increases intracellular pH (pHI) from 6.92 to 7.70 in fully grown prophase-blocked oocytes of the starfish Marthasterias glacialis, like external application of ammonia and other weak bases (M. Doree, K. Sano, and H. Kanatani, 1982, Dev. Biol.90, 13–17), inhibited meiosis reinitiation induced by 1-methyladenine (1-MeAde) or dithiothreitol (DTT), a mimetic of the hormone. Oocytes could be released from inhibition by raising the concentration of hormone or of its mimetic. Increasing pHI to 7.70 neither inhibited nor delayed meiosis reinitiation when pH was clamped after the end of the hormone-dependent period, the period during which 1-MeAde is required in the external medium for meiosis to occur, whereas it blocked the action of the hormone at low concentration when performed before the end of the hormone-dependent period. When hormone concentration was higher, germinal vesicle breakdown (GVBD) occurred, but duration of the hormone-dependent period was increased. Delay introduced by alkalinization for oocytes to reach GVBD after 1-MeAde addition was smaller at high than at medium concentrations of the hormone. Increasing pHI did not inhibit action of MPF, the cytoplasmic maturation factor which induces GVBD and the subsequent process of meiotic maturation following hormonal treatment of prophase-blocked oocytes.     

Hormone-induced phosphorylation of a 16000 Dalton polypeptide following meiosis reinitiation in starfish oocytes

In vitro phosphorylation of endogenous proteins is increased in homogenates prepared from 1-methyladenine-treated starfish oocytes when compared with control oocytes, although addition of the hormone to homogenates from control oocytes has no such effect. Following hormonal stimulation the best endogenous substrate is by far a 16 000 dalton (D) protein, the content of which also seems to increase, perhaps through proteolysis of a 21 000 D precursor. cAMP-dependent protein kinases are not involved in either basal or hormone-stimulated phosphorylations, as demonstrated by the lack of effect of either cAMP or of the heat-stable inhibitor of cAMP-dependent protein kinase on the extent of phosphorylation of individual endogenous substrates. Addition of 0.1 mM Ca²⁺ decreases to some extent the protein kinase activity in starfish homogenates and specifically suppresses the phosphorylation of a 40 000 D membrane protein. Starfish oocytes appear to contain myosin light chain kinase activity, as shown by the ability of homogenates to catalyse phosphorylation of exogenous 20 000 D myosin light chains.     

Aorta elastin turnover in normal and hypercholesterolemic Japanese quail

The turnover and degradation of mature elastin from the aortae of Japanese quail were estimated following with l-[U-¹⁴C]lysine by measuring the changes in specific activity of l-[U-¹⁴C]lysine and ¹⁴C-labelled desmosine and isodesmosine (crosslinking amino acids derived from lysyl residues) in elastin over a 39-week period. Only 5% of the variation in radioactivity could be attributed to changes in time. Therefore, it was concluded that the best estimates of mature elastin turnover are only quantifiable in years. Dietary cholesterol in amounts sifficient to induce plaque formation and fragmentation of the elastic lamina in the aorta did not significantly influence turnover time. It would appear that once the total pool of elastin in aorta is stabilized as mature fibers it is not subject to proteolysis or resynthesis of sufficient magnitude to result in measurable turnover.     

The metabolism of phenylmercury by the rat

The metabolism of [U-(14)C]phenylmercury acetate was studied in the rat. After a single subcutaneous dose a small proportion is excreted unchanged in urine, and a larger amount in bile with some resorption from the gut. The greater part of the dose is broken down in the tissues to yield inorganic mercury which is excreted mainly in faeces, and conjugates of phenol and quinol are excreted in urine. In experiments in vitro phenylmercury is broken down by liver homogenates to release inorganic mercury and benzene; this reaction is effected by the soluble, but not the microsomal, fraction and does not require NADPH or NADH. No elemental mercury is formed under these conditions. It is probable that this reaction occurs in vivo and the benzene produced is rapidly converted into phenol and quinol by microsomal enzymes.     

Transglutaminase-like activity in Chrysanthemum leaf explants cultivated in vitro in relation to cell growth and hormone treatment

In Chrysanthemum leaf explants cultivated in vitro the capacity to covalently link polyamines to protein substances exists. This plant enzyme activity shows some similarities with mammalian transglutaminases. In foliar explants cultured on a medium promoting bud or root formation increasing levels of transglutaminase-like activity occurred during the first days of culture when cell multiplication was rapid then the levels declined as the rate of cell division decreased and differentiation occurred. Undifferentiated callus exhibited low transglutaminase-like activity. Transglutaminase-like activity also increased in rapidly proliferating and growing organs (roots and buds initiated from the foliar explants) and decreased during maturity. The relationship among transglutaminases-like activity, cell division, bud and root formation is discussed.Abbreviations TGase transglutaminase - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Put putrescine - Spd spermidine     

Characterization of the pea ENOD12B gene and expression analyses of the two ENOD12 genes in nodule, stem and flower tissue

Summary The ENOD12 gene family in pea consists of two different members. The cDNA clone, pPsENOD12, represents the PsENOD12A gene. The second ENOD12 gene, PsENOD12B, was selected from a genomic library using pPsENOD12 as a probe and this gene was sequenced and characterized. The coding regions of the two genes are strikingly similar. Both encode proteins having a signal peptide sequence and a region with pentapeptide units rich in prolines. ENOD12A has a series of rather conserved repeating pentapeptide units, whereas in ENOD12B the number of pentapeptide units is less and these are less conserved. From the amino acid sequence it is obvious that the PsENOD12 genes encode proline-rich proteins which are closely related to proteins that have been identified as components of soybean cell walls (SbPRPs). Previously, Northern blot analyses had shown that ENOD12 genes are expressed in a tissues-pecific manner. A high expression level is found in Rhizobium-infected roots and in nodules, whereas expression in flower and stem is lower. This raised the question of which gene is expressed where and when. The availability of the sequences of both ENOD12 genes allowed us to analyse the expression of the two genes separately. Specific oligonucleotides were used to copy the ENOD12 mRNAs and to amplify the cDNAs in a polymerase chain reaction. It was demonstrated that in all the tissues containing ENOD12 mRNA, both genes PsENOD12A and PsENOD12B are transcribed and that the relative amounts of PsENOD12A and PsENOD12B mRNA within each tissue are more or less equal. Moreover, the expression pattern during infection and nodule development is the same for the two genes. These results show that two closely related genes have the same tissue-specific expression pattern and that the gene that we have isolated is an actively transcribed gene. The 2.7 kb genomic region that contains the PsENODI2B gene has a 41 pb nearly direct repeat in the 5 flanking region of the gene (between -1447 and -1153) and another 14 by direct repeat 3' downstream (between 550 and 626). The region between the AGGA box and the TATA box has a striking homology with the same region in SbPRP genes.