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1.
The data presented clearly suggest that relative amounts of mRNAs for elastins a, b and c are developmentally regulated in foetal-calf nuchal ligament and aorta and that this regulation is tissue-specific. In nuchal ligament, at earlier stages of foetal development, the relative amounts of mRNAs for elastins a and b are very low. After the foetal age of about 6 months the relative amount of mRNA for elastin b begins to increase. This is followed by an increase in the relative amount of mRNA for elastin a. In aorta, with increasing foetal age, the relative amounts of mRNAs for elastins b and c increase and decrease alternately. The relative amounts of mRNA for elastin a remain low, with only marginal increases with foetal age. A possible self-aggregation role of elastin a in elastogenesis is proposed. 相似文献
2.
Elastin mRNA levels during foetal development of sheep nuchal ligament and lung. Hybridization to complementary and cloned DNA 总被引:3,自引:0,他引:3
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J M Davidson S Shibahara C Boyd M L Mason P Tolstoshev R G Crystal 《The Biochemical journal》1984,220(3):653-663
Elastin mRNA levels were quantified in sheep nuchal ligament and lung during the latter half of foetal development with elastin-specific cDNA (complementary DNA) probes using both hybridization in solution (saturation analysis) and hybridization on a fixed support (Northern analysis). For the solution-hybridization studies, cDNA prepared from nuchal-ligament mRNA was enriched to 65% for elastin sequences by hybridizing it to its template at a R0t (mol X s X litre-1) value that included only the abundant class of mRNA sequences. Hybridization of this probe to RNA extracted from nuchal ligament between 70 and 138 days after conception demonstrated elastin sequences increased about 10-fold (from 0.047 to 0.438% of total RNA). In contrast, lung elastin mRNA levels increased only 3-fold (from 0.009 to 0.022% of total RNA) during the same period. Over this development period these values correspond to increases in the average number of elastin mRNA molecules from 950 to 20 000 molecules/ligament cell and from 130 to 330 molecules/lung cell. For Northern analysis, elastin mRNA was purified from near-term-sheep nuchal ligament on sucrose density gradients. Analysis of the translation products of this elastin mRNA showed that relative elastin precursor synthesis was at least 80% of total [3H]valine incorporation. The Mr of this elastin mRNA, determined by methylmercury-agarose-gel electrophoresis, was approx. 1.25 X 10(6). Northern hybridization of nuchal ligament and lung RNA to a [32P]cDNA probe, transcribed from this sucrose-gradient-purified elastin mRNA, confirmed the developmental changes in elastin mRNA levels detected by solution-hybridization techniques. The specificity of this method was confirmed by using a cloned elastin gene fragment. These studies demonstrate that elastin mRNA levels in organs such as nuchal ligament and lung increase with foetal development, but that there are significant differences in the average cellular elastin mRNA content of these two organs. 相似文献
3.
Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production 总被引:13,自引:7,他引:6
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《The Journal of cell biology》1981,90(2):332-338
Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis. 相似文献
4.
The release of soluble carbohydrates from isolated cell wall of maize (Zea mays L.) was investigated in the range of pH 1 to 8.5. The pH profile demonstrated two peaks, a broad peak at pH 6 due to enzymatic breakdown of β-glucan to monosaccharides (wall autolysis) and a sharp peak at pH 2.5 due to acid-mediated, nonenzymatic liberation of macromolecular β-glucan from the wall. The pH dependence of acid-induced growth and cell-wall extensibility of coleoptile segments closely agrees with the pH dependence of acid-mediated β-glucan solubilization in the isolated wall. However, there is no evidence that enzymatic or nonenzymatic β-glucan solubilization is involved in the mechanism of auxin-mediated growth. 相似文献
5.
Collagen heterogeneity and quantification in developing bovine nuchal ligament. 总被引:1,自引:1,他引:0
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The collagenous components were investigated in peptic digests of developing bovine nuchal ligament. Types I and III collagen were the major species isolated, but the presence of types IV, V and VI was also shown. Changes in the pepsin-susceptibility of nuchal ligament during foetal development were observed. CNBr-cleavage peptide analysis indicated that type I collagen became cross-linked rapidly, as evidenced by the lack of alpha 1(I)CB6. At present it is not clear if this decrease in pepsin-susceptibility is due to cross-linking of collagen, to increased deposition of elastin, or to both. Quantification of collagen types I and III was shown to depend on the method used. When pepsin-solubilized material was examined an apparent increase in type III collagen with respect to foetal age was observed, whereas when CNBr digests of intact ligament were examined a relatively constant amount of type III collagen (approx. 24%) was found. The constant amount of type III collagen observed during foetal development changed at birth and increased in mature nuchal ligament to represent approx. 45% of the total collagen. 相似文献
6.
M B?ni-Schnetzler W Scott S M Waugh E DiBella P F Pilch 《The Journal of biological chemistry》1987,262(17):8395-8401
Treatment of the soluble insulin receptor from human placenta with 1.25 mM dithiothreitol and 75 mM Tris at pH 8.5 results in complete reduction of interhalf disulfide bonds (class 1 disulfides) and dissociation of the tetrameric receptor into the dimeric alpha beta form. The alpha beta receptor halves exhibit a reduced affinity for insulin binding (B?ni-Schnetzler, M., Rubin, J. B., and Pilch, P. F. (1986) J. Biol. Chem. 261, 15281-15287). Kinetic experiments reveal that reduction of class 1 disulfides is a faster process than the loss of affinity for ligand, indicating that events subsequent to reduction of interhalf disulfides are responsible for the affinity change. We show that a third class of alpha subunit intrachain disulfides is more susceptible to reduction at pH 7.6 than at pH 8.5 and appears to form part of the ligand binding domain. Reduction of the intrachain disulfide bonds in this part of the alpha subunit leads to a loss of insulin binding. Modification of this putative binding domain by dithiothreitol can be minimized if reduction is carried out at pH 8.5. When the insulin receptor in placental membranes is reduced at pH 8.5, the receptor's affinity for insulin is not changed when binding is measured in the membrane. However, the Kd for insulin binding is reduced 10-fold when alpha beta receptor halves are subsequently solubilized. Scatchard analysis of insulin binding to reduced or intact receptors in the membrane and in soluble form together with sucrose density gradient analysis of soluble receptors suggests that alpha beta receptor halves remain associated in the membrane after reduction, but they are dissociated upon solubilization. We interpret these results to mean that the association of two ligand binding domains, 2 alpha beta receptor halves, is required for the formation of an insulin receptor with high affinity for ligand. 相似文献
7.
Terminal differentiation of nuchal ligament fibroblasts: characterization of synthetic properties and responsiveness to external stimuli 总被引:1,自引:0,他引:1
The temporal expression of elastogenesis is unique among connective tissues in that elastin production occurs primarily during late fetal and early neonatal periods and is essentially fully repressed once fiber assembly is completed. To test whether elastin synthesis in adult nuchal ligament fibroblasts is permanently repressed or whether the cells retain the ability to reinitiate production upon proper stimulation, we examined in adult ligament cells various parameters known to be involved in the regulation of elastin production. Elastin synthetic capacity, as determined by the levels of steady-state tropoelastin mRNA, of adult tissue was significantly decreased relative to fetal tissue. Likewise, fibroblasts grown from explants of adult ligament had about a fourfold decrease in elastin production and elastin-specific mRNA levels. On the other hand, adult cells were similar to fetal ligament cells in that they were sensitive to glucocorticoid stimulation and demonstrated chemotactic responsiveness to elastin peptides. Since our previous studies have shown that the extracellular matrix (ECM) plays an important role in influencing elastin phenotypic expression, fetal and adult fibroblasts were grown on slices of nonviable adult ligament to test if repression of elastin production was directed by factors in ECM of adult tissues. No change in elastin synthesis was detected with either cell type grown on adult ligament, whereas both fetal and adult cells demonstrated increased elastin production in response to contact with fetal ligament. These results suggest that adult ligament ECM does not provide a metabolic signal to shut off the elastin gene and that adult cells remain responsive to external stimuli that may reinitiate high levels of elastin synthesis. 相似文献
8.
Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver 总被引:45,自引:28,他引:17
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1. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase were measured in foetal, newborn and adult rat liver extracts by a radiochemical assay involving the fixation of [14C]bicarbonate. 2. Pyruvate-carboxylase activity in both foetal and adult liver occurs mainly in mitochondrial and nuclear fractions, with about 10% of the activity in the cytoplasm. 3. Similar studies of the intracellular distribution of phosphoenolpyruvate carboxykinase show that more than 90% of the activity is in the cytoplasm. However, in the 17-day foetal liver about 90% of the activity is in mitochondria and nuclei. 4. Pyruvate-carboxylase activity in both particulate and soluble fractions is very low in the 17-day foetal liver and increases to near adult levels before birth. 5. Phosphoenolpyruvate-carboxykinase activity in the soluble cell fraction increases 25-fold in the first 2 days after birth. This same enzyme in the mitochondria has considerable activity in the foetal and adult liver and is lower in the newborn. 6. Kinetic and other studies on the properties of phosphoenolpyruvate carboxykinase have shown no differences between the soluble and mitochondrial enzymes. 7. It is suggested that the appearance of the soluble phosphoenolpyruvate carboxykinase at birth initiates the rapid increase in overall gluconeogenesis at this stage. 相似文献
9.
Recombinant eglin c is a potent reversible inhibitor of human pancreatic elastase. At pH 7.4 and 25 degrees C, kass. = 7.3 x 10(5) M-1.s-1, kdiss. = 2.7 x 10(-4) s-1 and Ki = 3.7 x 10(-10) M. Stopped-flow kinetic indicate that the formation of the stable enzyme-inhibitor complex is not preceded by a fast pre-equilibrium complex or that the latter has a dissociation constant greater than 0.3 microM. The elastase-eglin c complex is much less stable at pH 5.0 and 25 degrees C, where kdiss. = 1.1 x 10(-2) s-1 and Ki = 7.3 x 10(-8) M. At pH 7.4 the activation energy for kass. is 43.9 kJ.mol-1 (10.5 kcal.mol-1). The kass. increases between pH 5.0 and 8.0 and remains essentially constant up to pH 9.0. This pH-dependence could not be described by a simple ionization curve. Both alpha 2-macroglobulin and alpha 1-proteinase inhibitor are able to dissociate the elastase-eglin c complex, as evidenced by measurement of the enzymic activity of alpha 2-macroglobulin-bound elastase or by polyacrylamide-gel electrophoresis of mixtures of alpha 1-proteinase inhibitor and elastase-eglin c complex. The rough estimate of kdiss. obtained with the alpha 2-macroglobulin dissociation experiment (1.6 x 10(-4) s-1) was of the same order of magnitude as the constant measured with the progress curve method. Eglin c strongly inhibits the solubilization of human aorta elastin by human pancreatic elastase. The extent of inhibition is the same whether elastase is added to a suspension of elastin and eglin c or whether elastase is preincubated with elastin for 3 min before addition of eglin c. However, the efficiency of the inhibitor sharply decreases if elastase is reacted with elastin for more prolonged periods. 相似文献
10.
Degradation of Kraft Indulin Lignin by Streptomyces viridosporus and Streptomyces badius 总被引:1,自引:0,他引:1
Crawford and collaborators have studied extensively the solubilization of lignocellulose by two Streptomyces species, S. badius and S. viridosporus. Using a condensed industrial lignin essentially devoid of carbohydrates, Indulin AT, as the sole source of carbon, similar results were obtained: (i) the growths of the bacteria were optimum at pH 7.5 to 8.5; (ii) yeast extract was a better source of nitrogen than NH(4)Cl; (iii) the products of the depolymerization of Indulin were soluble, acid-precipitable polymers. When d-glucose was added as a secondary carbon source, it was used preferentially and the production of acid-precipitable polymers began only after the complete depletion of the sugar. On the assumption that the degradation of Indulin was catalyzed by enzymes, proteins found in the culture media and soluble and insoluble intracellular proteins were incubated with Indulin at pH 7.0 at 37 degrees C. Proteins in all fractions from S. badius had ligninolytic activities which, with the exception of those in the intracellular soluble fraction, were increased in the presence of H(2)O(2). In S. viridosporus, both extra- and intracellular soluble activities were found which were not increased by H(2)O(2). The extracellular activity of S. viridosporus was not affected by heat, resisted partially an exposure to pH 1.0, and was completely destroyed by proteolysis. 相似文献
11.
Cleide Mara Rosa da Silva Rosa Maria Chura-Chambi Lennon Ramos Pereira Yraima Cordeiro Luís Carlos de Souza Ferreira Ligia Morganti 《BMC biotechnology》2018,18(1):78
Background
Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein.Results
Pressure-treatment (2.4?kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH?8.5, NS1 was refolded and formed soluble oligomers. High (up to 68?mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH?11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods.Conclusions
The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.12.
Elastin is an extracellular matrix protein found in adult and neonatal vasculature, lung, skin and connective tissue. It is secreted as tropoelastin, a soluble protein that is cross-linked in the tissue space to form an insoluble elastin matrix. Cross-linked elastin can be found in association with several microfibril-associated proteins including fibrillin-1, fibrillin-2 and fibulin-1 suggesting that these proteins contribute to elastic fiber assembly, structure or function. To date, the earliest reported elastin expression was in the conotruncal region of the developing avian heart at 3.5 days of gestation. Here we report that elastin expression begins at significantly earlier developmental stages. Using a novel immunolabeling method, the deposition of elastin, fibrillin-1 and -2 and fibulin-1 was analyzed in avian embryos at several time points during the first 2 days of development. Elastin was found at the midline associated with axial structures such as the notochord and somites at 23 h of development. Fibrillin-1 and -2 and fibulin-1 were also expressed at the embryonic midline at this stage with fibrillin-1 and fibulin-1 showing a high degree of colocalization with elastin in fibers surrounding midline structures. The expression of these genes was confirmed by conventional immunoblotting and mRNA detection methods. Our results demonstrate that elastin polypeptide deposition occurs much earlier than was previously appreciated. Furthermore, the results suggest that elastin deposition at the early embryonic midline is accompanied by the deposition and organization of a number of extracellular matrix polypeptides. These filamentous extracellular matrix structures may act to transduce or otherwise stabilize dynamic forces generated during embryogenesis. 相似文献
13.
The temperature-dependent swelling of elastin 总被引:1,自引:0,他引:1
J M Gosline 《Biopolymers》1978,17(3):697-707
It is suggested that the temperature-dependent swelling behavior of water-swollen elastin is due entirely to the interaction of the numerous nonpolar groups in the elastin protein wiht the aqueous swelling solvent (i.e., ahydrophobic interaction). Flory-Rehner theory for network swelling was used to test this hypothesis. Calculated values for the solvent–polymer interaction parameter, χ1, derived from swelling data indicate that water is a very poor solvent for elastin at all temperatures over the range 0–70° C. Comparison of the calculated χ1 values with theoretical values for the free energy of interaction of nonpolar solutes and water strongly suggests that the swelling behavior of elastin can be attributed quantitatively to hydrophobic interactions. The implications of these results for the structure and elastic mechanism of elastin are discussed. 相似文献
14.
Extracellular matrix-specific induction of elastogenic differentiation and maintenance of phenotypic stability in bovine ligament fibroblasts 总被引:3,自引:2,他引:1
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《The Journal of cell biology》1984,98(5):1804-1812
We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development. Undifferentiated ( nonelastin - producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae. ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at approximately 180 d of gestation) does not stimulate elastin production in undifferentiated cells. Matrix-induced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix. Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable. Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM. The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer. When grown on tissue culture plastic, ligament cells secrete greater than 80% of newly synthesized tropoelastin into the culture medium. When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine. 相似文献
15.
16.
Morrison HM Welgus HG Owen CA Stockley RA Campbell EJ 《Biochimica et biophysica acta》1999,1430(2):179-190
Solubilization of elastin by human leukocyte elastase (HLE) cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. We now report quantitative measurements of the binding and catalytic interaction between HLE and elastin permitted by analogy to receptor-ligand systems. Our results indicated that a limited and relatively constant number of enzyme binding sites were available on elastin, and that new sites became accessible as catalysis proceeded. The activation energies and solvent deuterium isotope effects were similar for catalysis of elastin and a soluble peptide substrate by HLE, yet the turnover number for HLE digestion of elastin was 200-2000-fold lower than that of HLE acting on soluble peptide substrates. Analysis of the binding of HLE to elastin at 0 degrees C, in the absence of significant catalytic activity, demonstrated two classes of binding sites (Kd=9.3x10(-9) M and 2.5x10(-7) M). The higher affinity sites accounted for only 6% of the total HLE binding capacity, but essentially all of the catalytic activity, and dissociation of HLE from these sites was minimal. Our studies suggest that interaction of HLE with elastin in vivo may be very persistent and permit progressive solubilization of this structurally important extracellular matrix component. 相似文献
17.
Interaction between human cathepsins K, L, and S and elastins: mechanism of elastinolysis and inhibition by macromolecular inhibitors 总被引:1,自引:0,他引:1
Novinec M Grass RN Stark WJ Turk V Baici A Lenarcic B 《The Journal of biological chemistry》2007,282(11):7893-7902
Proteolytic degradation of elastic fibers is associated with a broad spectrum of pathological conditions such as atherosclerosis and pulmonary emphysema. We have studied the interaction between elastins and human cysteine cathepsins K, L, and S, which are known to participate in elastinolytic activity in vivo. The enzymes showed distinctive preferences in degrading elastins from bovine neck ligament, aorta, and lung. Different susceptibility of these elastins to proteolysis was attributed to morphological differences observed by scanning electron microscopy. Kinetics of cathepsin binding to the insoluble substrate showed that the process occurs in two steps. The enzyme is initially adsorbed on the elastin surface in a nonproductive manner and then rearranges to form a catalytically competent complex. In contrast, soluble elastin is bound directly in a catalytically productive manner. Studies of enzyme partitioning between the phases showed that cathepsin K favors adsorption on elastin; cathepsin L prefers the aqueous environment, and cathepsin S is equally distributed among both phases. Our results suggest that elastinolysis by cysteine cathepsins proceeds in cycles of enzyme adsorption, binding of a susceptible peptide moiety, hydrolysis, and desorption. Alternatively, the enzyme may also form a new catalytic complex without prior desorption and re-adsorption. In both cases the active center of the enzymes remains at least partly accessible to inhibitors. Elastinolytic activity was readily abolished by cystatins, indicating that, unlike enzymes such as leukocyte elastase, pathological elastinolytic cysteine cathepsins might represent less problematic drug targets. In contrast, thyropins were relatively inefficient in preventing elastinolysis by cysteine cathepsins. 相似文献
18.
Staphylococcal acid phosphatase: extensive purification and characterization of the loosely bound enzyme 总被引:4,自引:0,他引:4
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Acid phosphatase of Staphylococcus aureus PS55 was eluted from the surface of these cells with 1.0 m KCl at pH 8.5 by gentle agitation at 25 C and was purified 44-fold (51% recovery) by two cycles of dialysis and gel filtration. The eluted enzyme which had a 280/260 (nm) absorbancy ratio of 0.71 required at least 0.5 m salt solution for solubilization; however, most of the purified product which had a 280/260 (nm) absorbancy ratio of 1.72 was soluble in dilute buffer solution [0.01 m tris(hydroxymethyl)aminomethane chloride, pH 8.5]. Purified acid phosphatase appeared homogeneous according to the criteria of gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. In a starch block, migration was toward the cathode at pH 8.0. Maximal activity occurred at pH 5.2 to 5.3 and salt concentration had little effect on phosphatase activity up to 1.0 m KCl or NaCl. Progressive loss of enzymatic acitivity occurred at higher salt concentrations. Molecular weight of purified acid phosphatase was estimated to be 58,000. 相似文献
19.
Dido Vassilacopoulou James A. Ripellino Nikolaos Tezapsidis Vivian Y. H. Hook Nikolaos K. Robakis 《Journal of neurochemistry》1995,64(5):2140-2146
Abstract: The amyloid β peptide (Aβ) of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APPs), which are considered type I transmembrane proteins. Here we report that the soluble fraction of isolated adrenal medullary chromaffin granules (CG), a model neuronal secretory vesicle system, contains an antigen that immunochemically and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from full-length APP. A truncated APP fragment with intact Aβ sequence was also detected in the soluble fraction of CG. In vitro experiments showed that full-length APP was solubilized from CG membranes at 37°C as a function of pH, with a peak of activity between pH 8.5 and pH 9.0. Solubilization of full-length APP was inhibited by several protease inhibitors, including aprotinin, cystatin, and iodoacetamide, by the divalent cations Ca2+ and Zn2+ , and by preheating of the membranes. These results are consistent with and suggest the involvement of an enzymatic mechanism in the solubilization of potentially amyloidogenic full-length APP. Production of Aβ from a transmembrane APP predicts a proteolytic cleavage within the lipid bilayer, a site relatively inaccessible to proteases. Thus, the detected soluble, potentially amyloidogenic, full-length APP may be a substrate for the proteases producing Aβ. The detection of soluble APP with intact Aβ sequence in secretory vesicles is consistent with the extracellular topology of amyloid depositions. 相似文献
20.
J A Foster E Bruenger L Rubin M Imberman H Kagan R Mecham C Franzblau 《Biopolymers》1976,15(5):833-841
Circular dichroic studies of a desmosine crosslinked peptide reveal a hitherto undescribed elastin spectrum possessing a weak negative band at 230–235 nm, a weak positive band at 215 nm, and a maximum negative band at 190 nm. The spectrum is sensitive to both pH and temperature displaying increased ellipticity of the 215-nm band at acidic pH and low temperature. The general shape of the spectrum and its behaviour toward temperature changes suggest the presence of an extended helical conformation. Susceptibility of insoluble elastin to digestion with chymotrypsin is increased tenfold at low temperature (4°C), supporting the contention that the conformational change of the type described above occurs in insoluble elastin. Such changes in conformation would result in increased availability of aromatic amino-acid resiudues to peptide bond cleavage. 相似文献