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1.
The neutral beta-N-acetylhexosaminidase (hexosaminidase C) from human brain was partially purified (separated from lysosomal beta-N-acetylhexosaminidases by chromatography on a Con A-Sepharose column). Hexosaminidase C was inhibited by medium-chain fatty acids (monocarboxylic acids with chain-length between C6 and C9), whereas shorter-chain monocarboxylic acids showed no inhibitory effect. Studies on the inhibition mechanism showed an irreversible and pH-dependent inhibition which progresses with time and which is not reversed by the removal of fatty acids (by Bio-Beads SM-2). Similar inhibitory effects were also obtained using Triton X-100 (but not with homologous alkylamines). These results suggest that the hexosaminidase C inactivation is related to the hydrophobic properties of the inhibitor which acts as a denaturing agent mainly at acidic pH. The possibility has been discussed that this inactivation effect of monocarboxylic acid on hexosaminidase C could constitute a molecular model of the toxicity of medium-chain-length fatty acids. 相似文献
2.
Differential inhibition of beta-hexosaminidase A and beta-hexosaminidase B by suramin 总被引:1,自引:0,他引:1
G Constantopoulos S Rees B G Cragg J A Barranger R O Brady 《Biochemical and biophysical research communications》1981,101(4):1345-1349
The trypanocidal drug suramin is a potent inhibitor of β-hexosaminidase A with a Ki of about 4.5 μM, and to a lesser extent of β-hexosaminidase B (Ki 31.5 μM). β-Hexosaminidase B remained active in the presence of 1.0 mM suramin whereas the activity of β-hexosaminidase A was completely inhibited at 0.1 mM. 相似文献
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Two distinct forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) were isolated from human liver mitochondria. They were referred to as HXM-A and HXM-B based on their order of elution from a DEAE-cellulose column. Activity of the two ligases was determined toward 15 different carboxylic acids. HXM-A represented 60-80% of the benzoate activity in the lysate, and kinetic analysis revealed that benzoate was the best substrate (highest V(max)/K(m)). The enzyme also had medium-chain fatty acid:CoA ligase activity. HXM-B had the majority of the hexanoate activity and hexanoate was its best substrate. It was, however, also active toward many xenobiotic carboxylic acids. Comparison of these two human XM-ligases with the previously characterized bovine XM-ligases indicated that they were kinetically distinct. When assayed with benzoic acid as substrate, both HXM-A and HXM-B had an absolute dependence on either Mg(2+) or Mn(2+) for activity. Further, addition of monovalent cation (K(+), Rb(+), or NH(4)(+)) stimulated HXM-A activity by >30-fold and HXM-B activity by 4-fold. For both forms, activity toward straight-chain fatty acids was stimulated less by K(+) than was activity toward benzoate or phenylacetate. A 60 kDa short-chain fatty acid:CoA ligase was also isolated. It had activity toward propionate and butyrate, but not acetate, hexanoate or benzoate. The K(m)(app) values were high but similar for propionate and butyrate (285 microM and 250 microM, respectively) but the V(max)(app) was nearly 6-fold greater with propionate as substrate. While the K(m) values are somewhat high, the enzyme is still more efficient with these substrates than either of the XM-ligases. 相似文献
5.
Determinants of intestinal mucosal uptake of short- and medium-chain fatty acids and alcohols 总被引:9,自引:0,他引:9
Uptake rates across the jejunal brush border have been measured for water-soluble fatty acids and alcohols and analyzed to determine the relative roles of the unstirred water layer and the lipid cell membrane as determinants of the intestinal absorptive process. Initial studies involving measurement of time courses of electrical transients developed across the intestine exposed to poorly permeant solute molecules showed no anomalous discrimination of probe molecules of different size or charge. This finding suggests that the diffusion barrier in the intestine can be considered as an unstirred water layer. Next, uptake rates of fatty acid were found to be linear with respect to concentration of the test solute, demonstrated no competitive inhibition or contralateral stimulation, had low temperature dependency, and were insensitive to metabolic inhibition, indicating that uptake proceeds by passive diffusion. Passive permeability coefficients, *P, varied from 22 +/- 1.4 to 395 +/- 9.2 nmoles.min(-1).100 mg(-1).mm(-1) for the saturated fatty acids 2:0 through 12:0 and from 119 +/- 3.3 to 581 +/- 45.2 for the saturated alcohols 6:0 through 10:0. Vigorous stirring of the bulk buffer solution enhanced *P values in direct proportion to chain length while the presence of bile acid micelles depressed apparent permeability coefficients in proportion to fatty acid chain length. These results demonstrate that uptake of short-chain fatty acid monomers is rate limited by the lipid cell membrane but diffusion through the unstirred water layer becomes increasingly rate limiting as the chain length increases. It is also possible to conclude from these data that diffusion through the unstirred water layer becomes totally rate limiting for uptake of long-chain fatty acid monomers of physiological importance. 相似文献
6.
beta-Hexosaminidase is a lysosomal hydrolase that is important in the metabolism of sphingoglycolipids. beta-Hexosaminidase B and beta-hexosaminidase A are the major isozymes in normal human tissue. beta-Hexosaminidase B is a homodimer of beta subunits, and beta-hexosaminidase A is a heterodimer composed of an alpha and a beta subunit. Crystals of beta-hexosaminidase B (M(r) 112,000) have been grown using the handling drop technique. They are elongated hexagonal prisms with maximum dimensions of 0.2 mm x 0.2 mm x 0.7 mm. The space group is P6(1)22 (or enantiomorph); the unit cell dimensions are a = b = 114.2 A, c = 402.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one dimer per asymmetric unit. Crystals diffract to 3.2 A resolution. 相似文献
7.
Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids. 总被引:5,自引:1,他引:4
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The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids. 相似文献
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Porcine liver aminopeptidase B[EC 3.4.11.6] is highly specific for hydrolysis of beta-naphthylamides of basic L-amino acids; the Km values for L-arginine beta-naphthylamide and L-lysine beta-naphthylamide were 0.035 and 0.12 mM, respectively. The enzyme was inhibited by various alpha-amino acids. Among basic amino acids, L-homoarginine and L-arginine were the most potent inhibitors, L-lysine and L-norarginine (alpha-amino-gamma-guanidinobutyric acid) being less inhibitory. Hydrophobic amino acids also inhibited the enzyme competitively. This suggests that there is a hydrophobic region that binds the side chain of the substrates or inhibitors in the specificity site of the enzyme. Studies on the inhibitions by L-arginine derivatives showed that blocking of the alpha-carboxyl or the alpha-amino group reduced the inhibitory effect of L-arginine. Porcine liver aminopeptidase B was not inhibited by puromycin, whereas bestatin inhibited the enzyme competitively with a Ki value of 1.4 X 10(-8) M. This enzyme had no kinin-converting activity. 相似文献
10.
Sven Wolfgang Sauer Juergen G. Okun Georg F. Hoffmann Stefan Koelker Marina A. Morath 《BBA》2008,1777(10):1276-1282
Accumulation of organic acids as well as their CoA and carnitine esters in tissues and body fluids is a common finding in organic acidurias, beta-oxidation defects, Reye syndrome, and Jamaican vomiting sickness. Pathomechanistic approaches for these disorders have been often focused on the effect of accumulating organic acids on mitochondrial energy metabolism, whereas little is known about the pathophysiologic role of short- and medium-chain acyl-CoAs and acylcarnitines. Therefore, we investigated the impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs on central components of mitochondrial energy metabolism, namely alpha-ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex, and single enzyme complexes I-V of respiratory chain. Although at varying degree, all acyl-CoAs had an inhibitory effect on pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity. Effect sizes were critically dependent on chain length and number of functional groups. Unexpectedly, octanoyl-CoA was shown to inhibit complex III. The inhibition was noncompetitive regarding reduced ubiquinone and uncompetitive regarding cytochrome c. In addition, octanoyl-CoA caused a blue shift in the gamma band of the absorption spectrum of reduced complex III. This effect may play a role in the pathogenesis of medium-chain and multiple acyl-CoA dehydrogenase deficiency, Reye syndrome, and Jamaican vomiting sickness which are inherited and acquired conditions of intracellular accumulation of octanoyl-CoA. 相似文献
11.
Hydrolysis of Tay-Sachs ganglioside by beta-hexosaminidase A of human liver and urine 总被引:6,自引:0,他引:6
Y T Li M Y Mazzotta C C Wan R Orth S C Li 《The Journal of biological chemistry》1973,248(21):7512-7515
12.
Sauer SW Okun JG Hoffmann GF Koelker S Morath MA 《Biochimica et biophysica acta》2008,1777(10):1276-1282
Accumulation of organic acids as well as their CoA and carnitine esters in tissues and body fluids is a common finding in organic acidurias, beta-oxidation defects, Reye syndrome, and Jamaican vomiting sickness. Pathomechanistic approaches for these disorders have been often focused on the effect of accumulating organic acids on mitochondrial energy metabolism, whereas little is known about the pathophysiologic role of short- and medium-chain acyl-CoAs and acylcarnitines. Therefore, we investigated the impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs on central components of mitochondrial energy metabolism, namely alpha-ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex, and single enzyme complexes I-V of respiratory chain. Although at varying degree, all acyl-CoAs had an inhibitory effect on pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity. Effect sizes were critically dependent on chain length and number of functional groups. Unexpectedly, octanoyl-CoA was shown to inhibit complex III. The inhibition was noncompetitive regarding reduced ubiquinone and uncompetitive regarding cytochrome c. In addition, octanoyl-CoA caused a blue shift in the gamma band of the absorption spectrum of reduced complex III. This effect may play a role in the pathogenesis of medium-chain and multiple acyl-CoA dehydrogenase deficiency, Reye syndrome, and Jamaican vomiting sickness which are inherited and acquired conditions of intracellular accumulation of octanoyl-CoA. 相似文献
13.
Summary A suite of sixteen monocarboxylic acids having carbon numbers 2 to 7, formed by the Miller-Urey spark discharge process, were identified and quantified by gas chromatography and mass fragmentography using a deuterium spiking technique. The molar concentration and isomeric distribution of these laboratory synthesized monocarboxylic acids are compared to those previously reported for the Murchison meteorite. They show similar trends, namely, decreasing molar concentration with increasing molecular weight, and, the ratio of normal/branched isomers tend toward smaller with increasing carbon numbers. 相似文献
14.
Presence of an atypical thermolabile species of beta-hexosaminidase B in metastatic-tumour tissue of human liver.
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An atypical thermolabile species of Hex B (hexosaminidase B) has been found in metastatic-tumour sites of human liver which has a thermostability curve similar to that of Hex A (hexosaminidase A), which is present in decreased amounts relative to the Hex A isoenzyme, and which exhibits decreased relative activity at acidic pH values (2.6-3.6) when compared with control-liver Hex B. This atypical Hex B isoenzyme has a normal apparent Michaelis constant (0.6 mM) for 4-methylumbelliferyl 2-deoxy-2-acetamido-beta-D-glucopyranoside. The presence of this atypical Hex B suggests that variant beta-chains are being produced in metastatic-tumour tissue. 相似文献
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The mechanism of inhibition of aminopeptidase M by bile acids was analyzed by application of the specific velocity plot that was introduced by Baici [Eur. J. Biochem. 119, 9-14 (1981)]. Kinetic studies with three bile acids (cholic acid, deoxycholic acid, and chenodeoxycholic acid) and three substrates (Leu-Met, Leu-Gly, and Leu-pNA) showed that the inhibition constants Ki for the bile acids were appreciably different from each other, but that the Ki for each was not affected by the substrates used, being 0.89-1.03 mM for cholic acid, 0.42-0.66 mM for deoxycholic acid, and 0.24-0.31 mM for chenodeoxycholic acid. The values of the kinetic coefficient alpha [(apparent Ks in the presence of inhibitor)/Ks] for cholic acid with Leu-Met and Leu-Gly were 9.0 and 2.5, respectively. These values were very similar to those for chenodeoxycholic acid (7.0 and 2.7) but smaller than those for deoxycholic acid (21 and 11). The values of the other kinetic coefficient beta [(apparent kp in the presence of inhibitor)/kp] were 0 except in the case of the combinations of Leu-Gly with cholic acid (0.33) and Leu-Gly with chenodeoxycholic acid (0.13). On the basis of these kinetic parameters, the inhibitions by bile acids were classified into 4 types: competitive-noncompetitive linear mixed type (1 less than alpha less than infinity, beta = 0), noncompetitive-uncompetitive linear mixed type (0 less than alpha less than 1, beta = 0), pure noncompetitive type (alpha = 1, beta = 0), and hyperbolic mixed type (1 less than alpha less than infinity, 0 less than beta less than 1).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
The uptake of beta-hexosaminidase (EC 3.2.1.30) in nonparenchymal liver cells (i.e. endothelial and Kupffer's cells) and peritoneal macrophages has been determined by an enzymatic assay. A considerable uptake was noted in nonparenchymal liver cells, whereas no measurable uptake was seen in peritoneal macrophages. The endothelial cells were more active in the uptake of beta-hexosaminidase than were the Kupffer's cells. The uptake of beta-hexosaminidase by nonparenchymal liver cells showed saturation kinetics and was competitively inhibited by mannan. These findings support the concept that a cell surface receptor on nonparenchymal liver cells mediates uptake of beta-hexosaminidase and suggests a difference in the receptor mechanisms on liver and peritoneal macrophages. 相似文献
18.
beta-hexosaminidase B is an enzyme that is involved in the degradation of glycolipids and glycans in the lysosome. Mutation in the HEXB gene lead to Sandhoff disease, a glycolipid storage disorder characterized by severe neurodegeneration. So far, little structural information on the protein is available. Here, the complete analysis of the disulfide bond pattern of the protein is described for the first time. Additionally, the structures of the N-glycans are analyzed for the native human protein and for recombinant protein expressed in SF21 cells. For the analysis of the disulfide bond structure, the protein was proteolytically digested and the resulting peptides were analyzed by MALDI-MS. The analysis revealed three disulfide bonds (C91-C137; C309-C360; C534-C551) and a free cysteine (C487). The analysis of the N-glycosylation was performed by tryptic digestion of the protein, isolation of glycopeptides by lectin chromatography and mass measurement before and after enzymatic deglycosylation. Carbohydrate structures were calculated from the mass difference between glycosylated and deglycosylated peptide. For beta-hexosaminidase B from human placenta, four N-glycans were identified and analyzed, whereas the recombinant protein expressed in SF21 cells carried only three glycans. In both cases the glycosylation belongs to the mannose-core- or high-mannose-type, and some carbohydrate structures are fucosylated. 相似文献
19.
Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells 总被引:3,自引:0,他引:3
Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1. 相似文献
20.
Epithelial cells were isolated from the undifferentiated mammary glands of mature virgin female rats, and their lipogenic characteristics were studied. These cells synthesized predominantly medium-chain fatty acids, albeit at a low rate. In contrast, whole tissue from mammary glands of virgin rats synthesized predominantly long-chain fatty acids at a relatively higher rate, indicating that the lipogenic activity is dominated by the adipocyte component of the gland. Enzyme assays revealed that thioesterase II, the enzyme which regulates production of medium-chain fatty acids by the fatty acid synthetase, was present at a high activity in the undifferentiated mammary epithelial cells of virgin rats. Immunohistochemical studies confirmed this observation and showed that the regulatory enzyme was present exclusively in the epithelial cells lining the alveolar and ductal elements of the undifferentiated gland. This study demonstrates that the potential to elaborate tissue-specific medium-chain fatty acids is already expressed in the undifferentiated tissue of virgin rats and is not acquired as a result of the differentiation associated with the lactogenic phase of development. In this species mammary epithelial cells apparently synthesize predominantly medium-chain fatty acids at all stages of development, and only the overall rate of synthesis is increased on induction of the fatty acid synthetase during lactogenesis. 相似文献