共查询到20条相似文献,搜索用时 578 毫秒
1.
《Free radical research》2013,47(5-6):307-313
Oxidation-induced increase of the net negative charge on low-density lipoprotein was studied by electro-phoretic mobility and by electron paramagnetic resonance. The negative-charge increase is associated not only with neutralization of the lysine residues of apoprotein B, but also with the exposition of the excessive negatively charged residues on the lipoprotein surface. The accumulation of the negatively charged residues is believed to be brought about by the conformational change of apoprotein B, triggered by neutralization of lysines and cleavage of peptide bonds. Alternatively, reactive oxygen species could also convert histidine to aspartic acid and proline to glutamic acid. 相似文献
2.
1. Different lipoprotein density fractions from pig serum were isolated by phosphotungstate precipitation followed by purification in the preparative ultra-centrifuge. 2. The protein part of very low density lipoproteins was composed of approximately 52 percent lipoprotein B apoprotein and the rest of lipoprotein C II apoprotein and other as yet unidentified peptides. 3. The protein moiety of low density lipoproteins consisted primarily of lipoprotein B apoprotein (over 95 percent); the amino acid compositions of lipoprotein B apoprotein of very low and low density lipoproteins were practically identical. 4. The predominant polypeptide of pig serum high density lipoproteins exhibited an amino acid composition and a molecular weight very similar to human liprotein A I apoprotein. In contrast to human lipoprotein A I apoprotein, the apoprotein from pigs was found to release leucine first followed by alanine, threonine, and lysine upon incubation with carboxypeptidase A. 5. In pig serum the major lipoprotein C apoprotein was found to be a polypeptide similar in amino acid composition to lipoprotein C II apoprotein from human serum. The molecular weight of this polypeptide is approximately 8000. Incubation experiments with carboxypeptidase A indicate serine to be the most likely C-terminal amino acid. 相似文献
3.
G Pifat J Brnjas-Kraljevi? G Jürgens C M Herak-Kramberger J N Herak 《Chemistry and physics of lipids》1992,63(3):159-167
The EPR technique with paramagnetic Mn(II) ions has been used to probe the negatively charged sites on the surface of modified low-density lipoprotein (LDL). LDL modified in five different ways exhibited increased binding capacity for divalent cations. Enhanced binding is caused by the increase in the number of 'strong' binding sites. The 'strong' sites have been identified to be the aspartic acid and/or glutamic acid carboxyl residues and the 'weak' sites are zwitter-ionic phospholipids. In native LDL the negative groups make 'bonds' with the positive lysyl residues, thus stabilizing the structure. Any deprotonation or modification of the lysine amino groups makes the LDL structure more loose and the amino acid carboxyl groups accessible to divalent cations. 相似文献
4.
J B Ragland P D Bertram S M Sabesin 《Biochemical and biophysical research communications》1978,80(1):81-88
A high density lipoprotein fraction accumulates in the plasma of patients with alcoholic hepatitis when a severe lecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for lecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism. 相似文献
5.
Apoproteins of hen's egg yolk very low density lipoprotein has been separated by Sephadex G-200 gel filtration in 0.5% sodium dodecyl sulfate into three categories of proteins termed apoprotein A, apoprotein B and apoprotein C. Apoprotein A fraction consists of several aggregated proteins (linked possibly by -S-S- bridges) as shown by acrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Apoprotein B contains two major protein components, B1 and B2, with molecular weights of 78 000 and 64 000, respectively, and two minor proteins components. Apoprotein C was obtained in a pure form as a low molecular weight, -S-S- linked dimer protein and accounted for about 30% of the total protein. In the monomeric form, apoprotein C has a molecular weight of 9400. Apoprotein A and apoprotein B have similar amino acid composition, except in isoleucine content which is over two times in apoprotein B as compared to apoprotein A. Apoprotein C lacks histidine and is richer in arginine than apoproteins A or B. Apoprotein C has lysine as N-terminal, while apoproteins A and B have predominantly arginine as the N-terminal amino acid. All the three fractions contain carbohydrate residues, apoprotein B being the richest in carbohydrate content. Cold-stored apoproteins A forms a clear gel when dispersed in 0.5% sodium dodecyl sulfate at concentration of above 2 mg/ml, while apoprotein B forms a gel only above 10 mg/ml. Apoprotein C, even at 35 mg/ml, forms a clear solution with no tendency to gel. 相似文献
6.
U Saxena A Nagpurkar P J Dolphin S Mookerjea 《The Journal of biological chemistry》1987,262(7):3011-3016
Rat serum phosphorylcholine-binding protein (PCBP), a member of the pentraxin family of proteins, was previously shown to bind multilamellar liposomes prepared with egg phosphatidylcholine and lysophosphatidylcholine. The results suggested that the phosphorylcholine groups on the surface of liposomes play an important role in the binding process (Nagpurkar, A., Saxena, U., and Mookerjea, S. (1983) J. Biol Chem. 258, 10518-10523). A study on the binding of human plasma lipoproteins to PCBP immobilized on Sepharose has now been initiated. Very low density lipoproteins were partially bound to a Sepharose-PCBP column, and the bound fraction contained higher concentrations of apoprotein B and E. All the low density lipoproteins applied were bound to the column. In the case of high density lipoproteins, only a small fraction was retained on the column (based on protein analysis), and that bound fraction contained all the apoprotein E and Lp(a) lipoprotein. The binding of very low, low, and high density lipoproteins to Sepharose-PCBP was Ca2+-dependent, and the bound lipoproteins were quantitatively eluted by a phosphorylcholine gradient. Apoprotein B and E were also bound when whole human plasma was applied to Sepharose-PCBP. The effect of selective modification of lysine residues by acetoacetylation and of arginine residues by cyclohexanedione on the binding of low density lipoproteins to Sepharose-PCBP was examined. Modification of arginyl residues resulted in marked reduction of binding, whereas modification of lysine had no effect. Removal of sialic acid from PCBP also had no effect on the binding of low density lipoproteins to immobilized-desialylated PCBP column. The preferential binding of apoprotein B- and E-containing lipoproteins to Sepharose-PCBP indicates a possible physiological role of PCBP and other similar circulating phosphorylcholine-binding proteins of the pentraxin family in lipoprotein metabolism. 相似文献
7.
Dmitri Beglov Chang‐Jin Lee Alfredo De Biasio Dima Kozakov Ryan Brenke Sandor Vajda Natalia Beglova 《Proteins》2009,77(4):940-949
The interactions of β2 glycoprotein I (B2GPI) with the receptors of the low‐density lipoprotein receptor (LDLR) family are implicated in the clearance of negatively charged phospholipids and apoptotic cells and, in the presence of autoimmune anti‐B2GPI antibodies, in cell activation, which might play a role in the pathology of antiphospholipid syndrome (APS). The ligand‐binding domains of the lipoprotein receptors consist of multiple homologous LA modules connected by flexible linkers. In this study, we investigated at the atomic level the features of the LA modules required for binding to B2GPI. To compare the binding interface in B2GPI/LA complex to that observed in the high‐resolution co‐crystal structure of the receptor associated protein (RAP) with a pair of LA modules 3 and 4 from the LDLR, we used LA4 in our studies. Using solution NMR spectroscopy, we found that LA4 interacts with B2GPI and the binding site for B2GPI on the 15N‐labeled LA4 is formed by the calcium coordinating residues of the LA module. We built a model for the complex between domain V of B2GPI (B2GPI‐DV) and LA4 without introducing any experimentally derived constraints into the docking procedure. Our model, which is in the agreement with the NMR data, suggests that the binding interface of B2GPI for the lipoprotein receptors is centered at three lysine residues of B2GPI‐DV, Lys 308, Lys 282, and Lys317. Proteins 2009. © 2009 Wiley‐Liss, Inc. 相似文献
8.
Inactivation of potassium channels plays an important role in shaping the electrical signalling properties of nerve and muscle cells. While it has been assumed that the rapid inactivation of the Kv1.4 channel is controlled by a “ball and chain” inactivation mechanism, the chain structure of the channel has not been well defined. Here, by conducting electrophysiological studies on variants containing mutations of the positively charged and negatively charged segments of the NH2-terminal of the channel protein, we show that neutralization or deletion of the positively charged segment (residues 83-98) significantly slowed the inactivation process. Replacement of this positively charged segment with the negatively charged segment (residues 123-137), and vice versa, so that both segments were simultaneously positively or negatively charged, also slowed the inactivation process. Furthermore, the inactivation process was not changed when the positively charged and the negatively charged segments were interchanged. In contrast, the voltage dependence of activation and inactivation of the channels was not significantly altered by these mutants. These results indicate that the electrostatic interaction between the positively and negatively charged segments plays a critical role in the inactivation process of the Kv1.4 channel. Taken together, we propose that the electrostatic interaction accelerates the inactivation of the Kv1.4 channel by making it easier for the inactivation ball to access its binding site. 相似文献
9.
Removal of acidic residues of the prodomain of PCSK9 increases its activity towards the LDL receptor
Holla ØL Laerdahl JK Strøm TB Tveten K Cameron J Berge KE Leren TP 《Biochemical and biophysical research communications》2011,(2):43054-238
Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and mediates intracellular degradation of the LDLR. The amino-terminus of mature PCSK9, residues 31–53 of the prodomain, has an inhibitory effect on this function of PCSK9, but the underlying mechanism is not fully understood. In this study, we have identified two highly conserved negatively charged segments (residues 32–40 and 48–50, respectively) within this part of the prodomain and performed deletions and substitutions to study their importance for degradation of the LDLRs.Deletion of the acidic residues of the longest negatively charged segment increased PCSK9’s ability to degrade the LDLR by 31%, whereas a modest 8% increase was observed when these residues were mutated to uncharged amino acids. Thus, both the length and the charge of this part of the prodomain were important for its inhibitory effect. Deletion of the residues of the shorter second negatively charged segment only increased PCSK9’s activity by 8%. Substitution of the amino acids of both charged segments to uncharged residues increased PCSK9’s activity by 36%. These findings indicate that the inhibitory effect of residues 31–53 of the prodomain is due to the negative charge of this segment. The underlying mechanism could involve the binding of this peptide segment to positively charged structures which are important for PCSK9’s activity. One possible candidate could be the histidine-rich C-terminal domain of PCSK9. 相似文献
10.
Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) containing various amounts of LPS were prepared in vitro. The 31P-NMR spectra showed that in the LDL-LPS complexes as well as in native LDL all phosphate groups of phospholipids are accessible to the paramagnetic shift reagent, Pr3+. Besides, the low frequency mobility of phospholipid phosphates in the complex is diminished. It was supposed that the phospholipid molecules in the LDL/LPS complex as in native LDL form a monolayer structure on the surface of LDL. The intrinsic fluorescence spectra of tryptophan residues of the apoprotein (apo B-100) revealed that the incorporation of LPS molecules into LDL particles is accompanied by minor changes in the conformation and orientation of the apo B molecule. As a result of these changes, certain fragments become exposed to a more hydrophilic environment and become more accessible to fluorescence quenchers. The use of charged (I-, Cs+) and uncharged (acrylamide) quenchers permitted to identify in the apo B molecule different tryptophan residues, some of which are localized in the vicinity of negatively charged groups, whereas others are neighbouring positively charged groups. It is suggested that the LPS molecules incorporated into LDL particle do not screen the apo B molecule to such an extent that it would hinder the LDL/LPS complex binding to apo B/E cellular receptors. 相似文献
11.
Changes in whole plasma and lipoprotien apoprotein concentrations were determined after a single injection of Triton WR 1339 into rats. Concentrations of apoproteins A-I (an activator of lecithin:cholesterol acyl transferase), arginine-rich apoprotein (ARP), and B apoprotein were measured by electroimmunoassay. The content of C-II apoprotein (an activaor of lipoprotein lipase) was estimated by the ability of plasma and lipoprotein fractions to promote hydrolysis of triglyceride in the presence of cow's milk lipase and also by isoelectric focusing on polyacrylamide gels. Apoproteins C-II and A-I were rapidly removed from high density lipoprotein (HDL) after Triton treatment and were recovered in the d 1.21 g/ml infranate fraction. A-I was then totally cleared from the plasma within 10--20 hr after injection. Arginine-rich apoprotein was removed from HDL and also partially cleared from the plasma. The rise in very low density lipoprotein (vldl) apoprotein that followed the removal of apoproteins from HDL was mostly antributed to the B apoprotein, although corresponding smaller increases were observed in VLDL ARP and C apoproteins. The triglyceride:cholesterol, triglyceride:protein, and B:C apoprotein ratios of VLDL more closely resembled nascent rather than plasma VLDL 10 hr after Triton injection. These studies suggest that the detergent may achieve its hyperlipidemic effct by disrupting HDL and thus removing the A-I and C-II proteins from a normal activating environment compirsing VLDL, HDL, and the enzymes. The possible involvement of intact HDL in VLDL catabolism is discussed in relation to other recent reports which also suggest that abnormalities of the VLDL-LDL system may be due to the absence of normal HDL. 相似文献
12.
Effect of alteration of charged residues at the N termini of signal peptides on protein export in Bacillus subtilis. 总被引:1,自引:0,他引:1
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The role of positively charged residues at the N termini of signal peptides in protein export has been studied in Bacillus subtilis. Bacillus signal peptides (alkaline protease [Apr] and neutral protease [Npr] from Bacillus amyloliquefaciens) were altered and fused to mature levansucrase (Lvs). The effects of the various alterations on the export of Lvs in B. subtilis were determined. The replacement of positively charged residues with neutral residues in both Apr and Npr signal peptides resulted in a slight defect in the export of Lvs from B. subtilis. Introduction of a negatively charged residue (aspartic acid) at the N terminus of Npr signal peptide blocked the export of Lvs. However, Apr signal peptide with a net charge of -3 (three aspartic acid residues) was still functional. 相似文献
13.
B J Clarke H C C?té D E Cool I Clark-Lewis H Saito R A Pixley R W Colman R T MacGillivray 《The Journal of biological chemistry》1989,264(19):11497-11502
We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation. 相似文献
14.
Wei-Jie Zhang Xin-Yi Wang Yu-Qi Guo Xiao Luo Xue-Juan Gao Xiao-Xia Shao Ya-Li Liu Zeng-Guang Xu Zhan-Yun Guo 《Amino acids》2014,46(5):1393-1402
Relaxin-3 is a newly identified insulin/relaxin superfamily peptide that plays a putative role in the regulation of food intake and stress response by activating its cognate G-protein-coupled receptor RXFP3. Relaxin-3 has three highly conserved arginine residues, B12Arg, B16Arg and B26Arg. We speculated that these positively charged arginines may interact with certain negatively charged residues of RXFP3. To test this hypothesis, we first replaced the negatively charged residues in the extracellular domain of RXFP3 with arginine, respectively. Receptor activation assays showed that arginine replacement of Glu141 or Asp145, especially Glu141, significantly decreased the sensitivity of RXFP3 to wild-type relaxin-3. In contrast, arginine replacement of other negatively charged extracellular residues had little effect. Thus, we deduced that Glu141 and Asp145, locating at the extracellular end of the second transmembrane domain, played a critical role in the interaction of RXFP3 with relaxin-3. To identify the ligand residues interacting with the negatively charged EXXXD motif of RXFP3, we replaced the three conserved arginines of relaxin-3 with negatively charged glutamate or aspartate, respectively. The mutant relaxin-3s retained the native structure, but their binding and activation potencies towards wild-type RXFP3 were decreased significantly. The compensatory effects of the mutant relaxin-3s towards mutant RXFP3s suggested two probable interaction pairs during ligand–receptor interaction: Glu141 of RXFP3 interacted with B26Arg of relaxin-3, meanwhile Asp145 of RXFP3 interacted with both B12Arg and B16Arg of relaxin-3. Based on these results, we proposed a relaxin-3/RXFP3 interaction model that shed new light on the interaction mechanism of the relaxin family peptides with their receptors. 相似文献
15.
The effects on slow inactivation (SI) of charge substitutions, neutralizations, and reversals were studied for the negatively charged residues D1309 and EE1314,15 surrounding the IFM motif in the DIII-DIV cytoplasmic linker - the putative fast inactivation particle - of human skeletal muscle voltage-gated sodium channel (hNa(V)1.4). Changing aspartate (D) at position 1309 to glutamate (E) (substitution) did not strongly affect SI, whereas charge neutralization to glutamine (Q) and charge reversal to arginine (R) right-shifted the midpoint of the steady-state SI curve. Charge neutralization (D-->Q) at position 1309 also reduced the apparent valence associated with SI. Glutamates (E) at positions 1314 and 1315 were similarly mutated. Charge reversal (EE-->RR) right-shifted the steady-state SI curve and both reversal and substitution (EE-->DD) reduced its apparent valence. Charge neutralization (EE-->QQ) and reversal decreased the maximum probability of SI. These mutations also had differential effects on the rate of SI onset and recovery. These results suggest that charged residues in the DIII-DIV linker may interact with structures that control SI. 相似文献
16.
The flavoenzyme d-amino acid oxidase from Rhodotorula gracilis is a homodimeric protein whose dimeric state has been proposed to occur as a result of (a) the electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3' of the other monomer, and (b) the interaction of residues (e.g. Trp243) belonging to the two monomers at the mixed interface region. The role of Trp243 was investigated by substituting it with either tyrosine or isoleucine: both substitutions were nondisruptive, as confirmed by the absence of significant changes in catalytic activity, but altered the tertiary structure (yielding a looser conformation) and decreased the stability towards temperature and denaturants. The change in conformation interferes both with the interaction of the coenzyme to the apoprotein moiety (although the kinetics of the apoprotein-FAD complex reconstitution process are similar between wild-type and mutant D-amino acid oxidases) and with the interaction between monomers. Our results indicate that, in the folded holoenzyme, Trp243 is situated at a position optimal for increasing the interactions between monomers by maximizing van der Waals interactions and by efficiently excluding solvent. 相似文献
17.
The binding of unmodified low density lipoproteins to the plasma membrane of fibroblasts was studied at the ultrastructural level. The bound low density lipoprotein was visualized by an indirect immunoperoxidase technique, with the use of an antiserum against apoprotein B. Immunoreactive regions representing bound apoprotein B were found on the plasma membrane, in indented regions with a diameter of 0.15–0.30 μm and a fuzzy coat on the cytoplasmic side. Fibroblasts from a patient homozygous for hyperlipoproteinaemia type IIa showed no immunoreactive material in the indented regions. The specific 125I-labelled low density lipoprotein binding to these homozygous fibroblasts was 7% compared to control fibroblasts. 相似文献
18.
T Sakurai S Kimura M Nakano H Kimura 《Biochemical and biophysical research communications》1991,177(1):433-439
This is the first observation for contributing to the glycation of low density lipoprotein (LDL) to oxidative modification of its own lipids and protein. Human plasma LDL was glycated by incubation with glucose (G-LDL). Glucose incorporated into apoprotein B was approximately 10 mol/mol of apoprotein (2.8% modification of lysine residues) and 84% of G-LDL was adsorbed on phenylboronate affinity column. G-LDL incubated with Fe3+ for 4 h caused a significantly higher level of lipid peroxidation than U-LDL (untreated with glucose), and a higher molecular weight protein was observed in apoprotein B on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), increasing with incubation period. Corresponding to change on SDS-PAGE, G-LDL exposed to Fe3+ moved faster than G-LDL per se or U-LDL to anode on agarose gel electrophoresis. The higher the Fe3+ concentration, the more lipid peroxidation was caused. Alpha-tocopherol or probucol suppressed the lipid peroxidation of G-LDL exposed to Fe3+. 相似文献
19.
Determination of the molecular weight of apoprotein subunits from low density lipoprotein by gel filtration 总被引:4,自引:0,他引:4
Another method has been developed for obtaining a soluble apoprotein from the low density lipoprotein (LDL) of human plasma in the density class 1.019 < d < 1.063. The approximate molecular weight of the apoprotein subunit from this lipoprotein density class was determined by gel filtration on Sephadex G-200 to be about 80,000. Both on gel filtration and analytical ultracentrifugation the soluble apoprotein showed one peak, but on cellulose acetate electrophoresis it showed two bands, which suggests two differently charged components. Because of the nature of the determination, the value of 80,000 probably represents an upper limit to the molecular weight of the LDL subunits. 相似文献
20.
Stephen R. Campion Elizabeth Elsasser Roger Chung 《Protein expression and purification》1997,10(3):331-339
AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export. 相似文献