共查询到20条相似文献,搜索用时 15 毫秒
1.
Sangaralingham SJ Heublein DM Grande JP Cataliotti A Rule AD McKie PM Martin FL Burnett JC 《American journal of physiology. Renal physiology》2011,301(5):F943-F952
Renal aging is characterized by structural changes in the kidney including fibrosis, which contributes to the increased risk of kidney and cardiac failure in the elderly. Studies involving healthy kidney donors demonstrated subclinical age-related nephropathy on renal biopsy that was not detected by standard diagnostic tests. Thus there is a high-priority need for novel noninvasive biomarkers to detect the presence of preclinical age-associated renal structural and functional changes. C-type natriuretic peptide (CNP) possesses renoprotective properties and is present in the kidney; however, its modulation during aging remains undefined. We assessed circulating and urinary CNP in a Fischer rat model of experimental aging and also determined renal structural and functional adaptations to the aging process. Histological and electron microscopic analysis demonstrated significant renal fibrosis, glomerular basement membrane thickening, and mesangial matrix expansion with aging. While plasma CNP levels progressively declined with aging, urinary CNP excretion increased, along with the ratio of urinary to plasma CNP, which preceded significant elevations in proteinuria and blood pressure. Also, CNP immunoreactivity was increased in the distal and proximal tubules in both the aging rat and aging human kidneys. Our findings provide evidence that urinary CNP and its ratio to plasma CNP may represent a novel biomarker for early age-mediated renal structural alterations, particularly fibrosis. Thus urinary CNP could potentially aid in identifying subjects with preclinical structural changes before the onset of symptoms and disease, allowing for the initiation of strategies designed to prevent the progression of chronic kidney disease particularly in the aging population. 相似文献
2.
T. J. Sprinkle † H. J. Sheedlo † T. B. Buxton J. P. Rissing‡ 《Journal of neurochemistry》1983,41(6):1664-1671
Abstract: Myelin proteins and the total Wolfgram protein fraction were isolated from the CNS of several mammalian species and characterized with rabbit anti-bovine 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) antisera after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose membranes. The corresponding CNP proteins cross-reacted across all species examined, suggesting that the CNP amino acid sequence was fairly well conserved in all six species. The same corresponding proteins were also identified immunochemically in the crude total Wolfgram protein fraction in the region of the W1 myelin protein, thus further supporting and extending two different previous reports indicating a relationship between CNP and the W1 protein. In addition to these CNS enzyme sources, peripheral nervous system CNP (rabbit and rat sciatic nerve) was also recognized by these same rabbit anti-bovine (CNS) CNP antisera. CNP was also detected in freshly isolated delipidated bovine oligodendrocyte membranes. These results suggest that rabbit anti-bovine CNP antisera may be of use in localization and structural studies of this enzyme in several different species and will permit clear identification of CNP in oligodendrocytes and their isolated membrane fractions. 相似文献
3.
High-resolution NMR studies of bacteriorhodopsin require the availability of the detergent-solubilized protein with both high concentration and small rotational correlation time. A procedure is described for the optimized preparation of such samples. Bacteriorhodopsin was first delipidated by detergent treatment of purple membrane under nonsolubilizing conditions for the protein. The delipidated aggregated protein could then be solubilized into monomers at concentration close to millimolar by selected detergents. The solubilizing detergent had an important effect on the rotational correlation time of the protein as shown by measuring in each case the temperature-dependent stability of the protein, the size of the detergent-protein complex, and the detergent viscosity. Consistently, a strong influence of the detergent was also found on spectral resolution in 13C NMR spectra of solubilized bacteriorhodopsin labeled with [1-13C]phenylalanine. Best resolution was obtained using n-dodecylmaltoside as detergent, with which relatively narrow well resolved 13C NMR resonances were observed at 50 degrees C. It is suggested that high-resolution NMR studies performed with this detergent may contribute to the structural resolution of bacteriorhodopsin. 相似文献
4.
William M. Schneider Yuefeng Tang S. Thangminlal Vaiphei Lili Mao Melissa Maglaqui Masayori Inouye Monica J. Roth Gaetano T. Montelione 《Journal of structural and functional genomics》2010,11(2):143-154
Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated
media. Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression
plasmids, can be condensed prior to protein expression, thereby providing high-quality 2H, 13C, 15N-enriched protein samples at 2.5–10% the cost of traditional methods. As an example of the value of such inexpensively-produced
perdeuterated proteins, we produced 2H, 13C, 15N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40× condensed phase media, and obtained NMR spectra suitable for 3D structure determination.
The cSPP system was also used to produce 2H, 13C, 15N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these
membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution.
This allows a membrane protein’s structural and functional properties to be characterized prior to reconstitution, or as a
probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a
standardized protocol for production of perdeuterated proteins using the cSPP system. The 10–40 fold reduction in costs of
fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated
proteins in spectroscopic and crystallographic studies. 相似文献
5.
Surface-exposed regions of membrane-bound myelin basic protein--the major extrinsic membrane protein of central nervous system myelin--have been implicated as possible antigenic sites in diseased myelin. With the goal of determining the extent and nature of these regions, we have prepared basic protein modified with 13CH3-enriched acetyl groups at 7 of its 13 lysine residues. The resulting protein was placed in a membrane environment and studied by NMR spectroscopy to determine the location and rates of molecular motion of the labeled side chains with respect to lipid bilayers of the membrane. When 13C NMR spectra were obtained of the acetylated protein bound to multilamellar vesicles prepared from dimyristoylphosphatidic acid in the gel state (T = 33 degrees C), conditions under which reduced motion in the lipid bilayer broadens methylene and methyl 13C resonances of the membrane beyond detection (i.e. greater than 75-100 Hz), line widths of membrane-bound protein were measured to be 7.8 Hz, an increase of 4 Hz versus free protein. A reduction of 25-30% in integrated intensity observed in protein acetyl resonances upon membrane interaction was shown to be attributable to a population of protein-aggregated liposomes whose resonances were similarly too broad to be observed. Thus, the epsilon-acetyllysyl probes distributed throughout the protein do not penetrate the dimyristoylphosphatidic acid bilayer, but must reside in the interstitial aqueous spaces at or between membrane surfaces. These findings suggest an overall surface accessibility of membrane-bound myelin basic protein and are therefore incompatible with a model for the protein involving membrane-embedded loops or regions of functional significance. 相似文献
6.
Cellular and Subcellular Distribution of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase and Its mRNA in the Rat Central Nervous System 总被引:7,自引:2,他引:5
Bruce D. Trapp Lise Bernier S. Brian Andrews † David R. Colman† 《Journal of neurochemistry》1988,51(3):859-868
The 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs) are closely related oligodendrocyte proteins whose in vivo function is unknown. To identify subcellular sites of CNP function, the distribution of CNP and CNP mRNA was determined in tissue sections from rats of various developmental ages. Our results indicate that CNP gene products were expressed exclusively by oligodendrocytes in the CNS. CNP mRNA was concentrated around oligodendrocyte perinuclear regions during all stages of myelination. Developmentally, initial detection of CNP mRNA closely paralleled initial detection of its translation products. In electron micrographs of immunostained ultrathin cryosections, CNP was associated with oligodendrocyte membranes during the earliest phase of axonal ensheathment. In more mature fibers, immunocytochemistry established that the CNPs are not major components of compact myelin but are concentrated within specific regions of the oligodendrocyte and myelin internode. These include (a) the plasma membrane of oligodendrocytes and their processes, (b) the periaxonal membrane and inner mesaxon, (c) the outer tongue process, (d) the paranodal myelin loops, and (e) the "incisure-like" membranes found in many larger CNS myelin sheaths. A cytoplasmic pool of CNP was also detected in oligodendrocyte perikarya and larger oligodendrocyte processes. CNP was also enriched in similar locations in myelinated fibers of the PNS. 相似文献
7.
Byungsu Kwon Daniel Tietze Paul B. White Shu Y. Liao Mei Hong 《Protein science : a publication of the Protein Society》2015,24(7):1087-1099
Solid‐state NMR‐based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly 13C‐labeled. A strategy to meet this challenge is chemical ligation combined with site‐specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water‐soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra‐membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly 13C‐labeled full‐length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid‐phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post‐AH cytoplasmic residues exhibit random‐coil chemical shifts, low bond order parameters, and a surface‐bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus‐mimetic membrane, indicating that the structure and dynamics of the post‐AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium‐sized membrane proteins to provide site‐specific structural constraints, which complement the information obtained from uniformly 13C, 15N‐labeled proteins. 相似文献
8.
Natriuretic peptide receptor-C signaling and regulation 总被引:10,自引:0,他引:10
Anand-Srivastava MB 《Peptides》2005,26(6):1044-1059
The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP(4-23), a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP(99-126) or C-ANP(4-23). In addition, C-ANP(4-23) also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP(4-23) and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed. 相似文献
9.
2',3'-Cyclic Nucleotide 3'-Phosphodiesterase Binds to Actin-Based Cytoskeletal Elements in an Isoprenylation-Independent Manner 总被引:2,自引:1,他引:1
Abstract: 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope-tagged CNP1 and a non-isoprenylated mutant CNP1. Using nonionic detergent extraction, drug-mediated cytoskeletal disruption, and coimmunoprecipitation with an anti-actin antibody, we show that CNP1 is associated with actin-based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c-H-ras, which is also modified by isoprenylation at its carboxyl-terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton. 相似文献
10.
2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is a protein found abundantly in the cytoplasmic compartments of CNS myelin. Two isoforms of this protein, CNP1 and CNP2, are detectable. They differ by a 20-amino acid extension exclusive to CNP2. Additionally, CNP2 is essentially the only isoform to be phosphorylated in vivo. In this study, we examine the phosphorylation of CNP2 in transfected cells. CNP2 was selectively expressed ectopically in 293T cells and labeled with 32P. Immunoprecipitation of labeled CNP2 and tryptic phosphopeptide mapping analyses identified serines 9 and 22 as the major sites of phosphorylation. Only serine 22 was phosphorylated initially in oligodendrocyte-enriched cultures of neonatal rat brain glial cells. However, 4beta-phorbol 12,13-dibutyrate (PDB) induced the phosphorylation of serine 9, thereby producing the same pattern seen in 293T cells. These results suggest that serine 9 is phosphorylated by a PDB-sensitive kinase, likely protein kinase C, and that serine 22 appears to be constitutively phosphorylated. 相似文献
11.
Auger M 《Current issues in molecular biology》2000,2(4):119-124
Nuclear magnetic resonance (NMR) spectroscopy, and particularly solid-state NMR spectroscopy, is a method of choice to study the structure and dynamics of both the lipid and the protein components of model and biological membranes. Different approaches have been developed to study these systems in which the restricted molecular motions result in broad NMR spectra. This contribution will first present an overview of the different techniques used to study lipid bilayers, namely 31p, 2H and 13C solid-state NMR spectroscopy. On the other hand, the study of the structure of membrane peptides and proteins is a rapidly growing field and several methods developed in the last two decades will be presented. These methods allow the investigation of protein systems for which structural information is often difficult to obtain by techniques such as X-ray diffraction and multidimensional solution NMR. 相似文献
12.
Klein MJ Grage SL Muhle-Goll C Bürck J Afonin S Ulrich AS 《Biochimica et biophysica acta》2012,1818(12):3025-3031
Tat signal peptides provide the key signature for proteins that get exported by the bacterial twin arginine translocase. We have characterized the structure of the PhoD signal peptide from Bacillus subtilis in suitable membrane-mimicking environments. High-resolution (13)C/(15)N NMR analysis in detergent micelles revealed a helical stretch in the signal peptide between positions 5 and 15, in good agreement with secondary structure prediction and circular dichroism results. This helix was found to be aligned parallel to the membrane surface according to oriented circular dichroism experiments carried out with planar lipid bilayers. The N-terminal α-helix exhibits a pronounced amphiphilic character, in contrast to the general view in the literature. So far, signal sequences had been supposed to consist of a positively charged N-terminal domain, followed by an α-helical hydrophobic segment, plus a C-terminal domain carrying the peptidase cleavage site. Based on our new structural insights, we propose a model for the folding and membrane interactions of the Tat signal sequence from PhoD. 相似文献
13.
Structural analysis of nanoscale self-assembled discoidal lipid bilayers by solid-state NMR spectroscopy
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Nanodiscs are an example of discoidal nanoscale self-assembled lipid/protein particles similar to nascent high-density lipoproteins, which reduce the risk of coronary artery disease. The major protein component of high-density lipoproteins is human apolipoprotein A-I, and the corresponding protein component of Nanodiscs is membrane scaffold protein 1 (MSP1), a 200-residue lipid-binding domain of human apolipoprotein A-I. Here we present magic-angle spinning (MAS) solid-state NMR studies of uniformly (13)C,(15)N-labeled MSP1 in polyethylene glycol precipitated Nanodiscs. Two-dimensional MAS (13)C-(13)C correlation spectra show excellent microscopic order of MSP1 in precipitated Nanodiscs. Secondary isotropic chemical shifts throughout the protein are consistent with a predominantly helical structure. Moreover, the backbone conformations of prolines derived from their (13)C chemical shifts are consistent with the molecular belt model but not the picket fence model of lipid-bound MSP1. Overall comparison of experimental spectra and (13)C chemical shifts predicted from several structural models also favors the belt model. Our study thus supports the belt model of Nanodisc structure and demonstrates the utility of MAS NMR to study the structure of high molecular weight lipid-protein complexes. 相似文献
14.
This report compares trafficking routes of a plasma membrane protein that was misfolded either during its synthesis or after it had reached the cell surface. A temperature-sensitive mutant form of the yeast alpha-factor pheromone receptor (ste2-3) was found to provide a model substrate for quality control of plasma membrane proteins. We show for the first time that a misfolded membrane protein is recognized at the cell surface and rapidly removed. When the ste2-3 mutant cells were cultured continuously at 34 degrees C, the mutant receptor protein (Ste2-3p) failed to accumulate at the plasma membrane and was degraded with a half-life of 4 min, compared with a half-life of 33 min for wild-type receptor protein (Ste2p). Degradation of both Ste2-3p and Ste2p required the vacuolar proteolytic activities controlled by the PEP4 gene. At 34 degrees C, Ste2-3p comigrated with glycosylated Ste2p on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that Ste2-3p enters the secretory pathway. Degradation of Ste2-3p did not require delivery to the plasma membrane as the sec1 mutation failed to block rapid turnover. Truncation of the C-terminal cytoplasmic domain of the mutant receptors did not permit accumulation at the plasma membrane; thus, the endocytic signals contained in this domain are unnecessary for intracellular retention. In the pep4 mutant, Ste2-3p accumulated as series of high-molecular-weight species, suggesting a potential role for ubiquitin in the elimination process. When ste2-3 mutant cells were cultured continuously at 22 degrees C, Ste2-3p accumulated in the plasma membrane. When the 22 degrees C culture was shifted to 34 degrees C, Ste2-3p was removed from the plasma membrane and degraded by a PEP4-dependent mechanism with a 24-min half-life; the wild-type Ste2p displayed a 72-min half-life. Thus, structural defects in Ste2-3p synthesized at 34 degrees C are recognized in transit to the plasma membrane, leading to rapid degradation, and Ste2-3p that is preassembled at the plasma membrane is also removed and degraded following a shift to 34 degrees C. 相似文献
15.
C. Stewart Gillespie Lise Bernier Peter J. Brophy David R. Colman 《Journal of neurochemistry》1990,54(2):656-661
We have investigated the site of synthesis of the 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin-associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane-bound receptor. 相似文献
16.
Jeffrey P. Bocock Stephanie Carmicle Egbert Madamba Ann H. Erickson 《Traffic (Copenhagen, Denmark)》2010,11(6):756-766
Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post‐translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12‐myristate 13‐acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full‐length protein and a membrane‐embedded C‐terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant‐negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress‐induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase‐active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane. 相似文献
17.
R Shapira W C Mobley S B Thiele M R Wilhelmi A Wallace R F Kibler 《Journal of neurochemistry》1978,30(4):735-744
Abstract— The enzyme 2′,3′-cyclic nucleotide-3′-phosphohydrolase (CNP) has been assayed in fractions from a continuous sucrose density gradient zonal centrifugation of rabbit brain homogenates. Basic protein (BP) was also assayed by a radioimmunomethod. Fractions were examined by SDS-polyacrylamide gel electrophoresis and by electron microscopy. These studies show that the major membrane fractions in the gradient differ greatly in the content of CNP and BP, and of high molecular weight proteins (HMW). The lightest membrane fractions contained numerous multilamellae, the highest content of BP and the lowest content of CNP and HMW, while the heaviest membrane fractions contained single membrane fragments and vesicles of unknown origin, the lowest content of BP and the highest content of CNP and HMW. The fraction containing the largest amount of membrane measured by turbidity, protein content, and water-washed dry weight contained only half the CNP specific activity of a denser fraction in the gradient. CNP specific activity in the lightest fractions was insignificant compared to that of denser fractions. Thus, we conclude that this enzyme may be absent from the typical multilamellar myelin structures but present in the single-membrane structures associated with myelin, such as the glial membrane and the paranodal segments of myelin adjacent to the axon. BP appears to occupy the opposite positions, highest in the multilamellae and lowest in the single-membrane structures of myelin. These studies do not exclude the possibility that CNP may not be bound to myelin membranes, but rather to a membrane of different origin. Evidence that this enzyme is a myelin-marker enzyme is circumstantial. Our evidence indicates the enzyme could be present either in a unique portion of myelin membranes or in another membrane structure. 相似文献
18.
2',3'-cyclic nucleotide-3'-phosphodiesterase in the central nervous system is fatty-acylated by thioester linkage 总被引:1,自引:0,他引:1
2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNP1 and CNP2 with Mr of 46,000 and 48,000, respectively) is the major enzyme of central nervous system myelin. It is associated with oligodendroglial plasma membrane and uncompacted myelin (myelin-like fraction), which are in contact with glial cytoplasm. Proteins of the myelin-like fraction were labeled with [3H]palmitic acid in brain slices from 17-day-old rats and immunoprecipitated with anti-CNP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material revealed intense acylation of CNP1 and CNP2, and radioactivity was released by hydroxylamine. Palmitic acid was covalently bound to CNP because radioactivity was not removed by extraction of immunoprecipitated CNP with organic solvent or by boiling in sodium dodecyl sulfate and dithiothreitol. However, treatment of immunoprecipitated CNP with (a) hydroxylamine-released palmitohydroxamate and palmitic acid, (b) sodium borohydride-released hexadecanol, and (c) methanolic-KOH-released methyl palmitate. Synthesis, acylation, or transport of CNP was not affected by monensin or colchicine. However, acylation of CNP was inhibited 24-32% by cycloheximide. These results provide conclusive evidence that CNP1 and CNP2 are fatty acid acylated with palmitate through a thioester linkage and is posttranslationally modified sometime after synthesis. 相似文献
19.
Previous studies have shown that human calcitonin (hCT) and its C-terminal fragment hCT(9-32) translocate in nasal epithelium. Moreover, hCT(9-32) was used as a carrier to internalize efficiently the green fluorescent protein, drugs, and plasmid DNA. To understand the mechanism of the membrane crossing process, we determined structural parameters of the carrier peptide hCT(9-32) in a membrane environment using solid-state NMR. For that purpose, we synthesized a multiply labeled hCT(9-32) peptide comprising four positions with fully (15)N- and (13)C-labeled amino acids. Multilamellar vesicle samples containing varying mixing ratios of hCT(9-32) and phospholipids found in the plasma membrane of nasal epithelium were prepared. The typical axially symmetric powder patterns of (31)P NMR spectra confirmed the presence of lamellar bilayers in our samples. The chemical shift anisotropy of the (31)P NMR spectra of the samples in the presence of hCT(9-32) is slightly reduced, revealing weak interaction of the peptide with the lipid headgroups. The peptide does not penetrate the lipid membrane as indicated by very similar (2)H NMR order parameters of the phospholipid fatty acid chains in the absence and presence of the carrier peptide. This membrane topology was confirmed by measurements of paramagnetic enhancement of relaxation rates. The conformation of hCT(9-32) was investigated by cross polarization magic angle spinning NMR methods. All peptide signals were resolved and fully assigned in two-dimensional proton-driven (13)C spin diffusion experiments. The isotropic chemical shifts of (13)CO, (13)Calpha, and (13)Cbeta provide information about the secondary structure of the carrier peptide. The conformation of hCT(9-32) was further corroborated by quantitative phi torsion angle measurements. Two monomeric structural models are consistent with the data: (i) a linear backbone conformation of hCT(9-32) and (ii) an antiparallel beta-sheet structure. These structures are maintained over a wide range of peptide:lipid mixing ratios. No direct indications for fibril formation of hCT(9-32) were found. Dipolar coupling measurements indicate rather high amplitudes of motion of the peptide. 相似文献