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1.
Marco Filice Marzia Marciello Lorena Betancor Alfonso V. Carrascosa Jose M. Guisan Gloria Fernandez‐Lorente 《Biotechnology progress》2011,27(4):961-968
Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20‐ to 25‐fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide‐activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl‐Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 相似文献
2.
A Santagostino G Giagnoni P Fumagalli D Pavesi E Torretta 《Biochemical and biophysical research communications》1982,104(2):577-582
The isoelectric point of the camel and the human β-endorphin, of the α-endorphin and the enkephalins were determined by analytical isoelectric focusing on 1 mm thin polyacrylamide gel slab. The difficulty of staining peptides as short as β-endorphin or smaller was overcomed using a modification of Bibring and Baxandall's or Faupel and Von Arx's staining method. The camel β-endorphin gives two bands having isoelectric point of 10.3 and 10.4, the human β-endorphin focus at pH 9.9, while α-endorphin, leu and met-enkephalin at pH 5.9, 5.5 and 5.45 respectively. The staining method described coupled with the isoelectric focusing seems to be fit for discriminating β-endorphin in a crude rat pituitary extract. 相似文献
3.
Bovine pancreatic trypsin inhibitor (BPTI, aprotinin) is a fifty-eight amino acid polypeptide, which is present together with related molecular isoforms in various bovine organs. In the present study these protease inhibitors were isolated from bovine kidney by affinity chromatography on immobilized trypsin and a subsequent FPLC step. Due to their electrophoretic, structural, and inhibitory properties, the inhibitors were strictly similar to the polypeptides identified previously in other bovine organs. Immunohistochemical experiments showed a widespread localization of these polypeptides in nephron epithelial cells (proximal and distal tubules, loop of Henle, collecting tubules). 相似文献
4.
Rita Businaro Evandro Fioretti Lorenzo Fumagalli Gennaro Citro Gabriella De Renzis Franca Ascoli 《The Histochemical journal》1988,20(4):187-193
Summary In addition to bovine pancreatic trypsin inhibitor (BPTI), three BPTI-related molecular forms (isoinhibitors I, II and III) were isolated from bovine lung by affinity chromatography on immobilized trypsin and subsequently purified by Fast Protein Liquid Chromatography. These inhibitors are identical to the isoinhibitors previously isolated from bovine spleen. Their localization in bovine lung was studied by immunohistochemical techniques, using two different immunoglobulin preparations, selectively recognizing BPTI or the other molecular forms.BPTI-related immunoreactivity was found to be restricted to isolated cells, often identified as mast cells by Toluidine Blue staining. In contrast, isoinhibitor-related immunoreactivity, which also occurs in the mast cells, is present in a number of other cell types. These types include: (i) the smooth muscle cells of different calibre vessels, (ii) the ciliated cells of the bronchial epithelium and the related mucus, and (iii) many cells at alveolar level.Comparison of these data with previous results obtained for bovine spleen suggest multiple physiological roles for these inhibitors. 相似文献
5.
Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa 下载免费PDF全文
Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane. 相似文献
6.
R. Fumagalli R. Caponi A. Corsini A. Brambilla M. Palmira F. Bernini U. Valcavi 《Prostaglandins & other lipid mediators》1985,29(3)
Experiments were designed to examine the possibility that nonprotein sulfhydryl groups of the gastric mucosa could participate in the protection of rat gastric mucosa by rosaprostol (the Na salt of 9-hydroxy-8,12 trans-19,20-bis-nor-prostanoic acid). Gastric mucosal lesions and the content of nonprotein sulfhydryls were evaluated after orally administered absolute ethanol. Pretreatment with rosaprostol by gavage prevented gastric lesions and reduced or prevented the decrease of mucosal nonprotein thiols. N-ethylmaleimide, a sulfhydryl blocker, worsened the ethanol-induced gastric lesions and lowered further the non protein thiols. Both variables were improved by the PG analogue and by PGE2. These results suggest a possible role of endogenous nonprotein sulfhydryl groups in the gastric protective effect of rosaprostol. 相似文献
7.
8.
THE EFFECT OF HYPOCHOLESTEREMIC AGENTS ON MYELINOGENESIS 总被引:4,自引:1,他引:3
Abstract— Three drugs known to inhibit biosynthesis of cholesterol, Clofibrate, 20, 25-diazacholesterol and AY-9944 were administered by stomach intubation to suckling rats. At weaning the rats were killed and subcellular fractions, including myelin, were prepared from the brains and spinal cords and analysed for sterol content. Central nervous tissue fractions from Clofibrate-treated rats showed some decrease in total sterols, but the sterol species were qualitatively normal. AY-9944 given to rats caused high amounts of 7-dehydro-cholesterol to accumulate in all brain and spinal cord fractions with the highest amounts (32–38 percent of total sterols) in myelin. In diazasterol-treated rats desmosterol reached 48 per cent of the sterols of myelin. A group of rats was allowed to survive after the final drug intake (21 days) and their brain and spinal cord sterol content followed up to 60 days. At 30 days the proportion of dehydrocholesterol or desmosterol comprised over half the total myelin sterol. By 60 days of age the 7-dehydrocholesterol had almost completely disappeared from all fractions while substantial amounts of desmosterol were retained in myelin. Myelination was retarded by treatment with AY-9944 and 20, 25-diazasterol, possibly by the limited amount of sterols available. The metabolism of the abnormal myelin constituents in drug-treated animals is discussed in relation to the molecular structure of the myelin membrane. 相似文献
9.
—The lipid composition of the central nervous system of some marine vertebrates and two mammalian species (rat and man) was analysed by one- and two-dimensional quantitative thin-layer chromatography, and the cerebroside fatty acids were analysed by gas chromatography. The concentrations of sphingomyelin and cerebrosides are higher in mammals than in fishes, while no significant differences are observed for other lipid classes. Furthermore, in mammals the ratio between hydroxy and normal fatty acids in the cerebrosides is much higher than in fishes. The cerebrosides of mammals contain more very long chain fatty acids than those of marine vertebrates. 相似文献
10.
Osvaldo Giorgi Marzia Orlandi Daniele Lecca Giuliana P. Serra Lei Zhang Maria G. Corda 《Journal of neurochemistry》1996,67(1):423-429
Abstract: The effects of GABA on the kinetics of tert -[35 S]butylbicyclophosphorothionate ([35 S]TBPS) binding to the convulsant site of GABAA receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [35 S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast ( t 1/2 , 11 min) and slow ( t 1/2 , 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µ M GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µ M GABA (dissociation started by adding 40 µ M picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µ M picrotoxin plus 2 µ M GABA). Taken together, these results suggest that (1) the closed state of GABAA receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS. 相似文献