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1.
1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed. 2. Of the compounds tested besides mannose, only those containing an alpha-linked mannosyl unit at the nonreducing position of their molecule were effective as acceptors. Monodeoxyanalogues of mannose as well as alpha-mannose phosphates did not serve as acceptors in the above reaction. 3. The structure of the product formed with mannose as acceptor was determined to be O-alpha-D-mannosyl-(1 leads to 2)-mannose; with alphaMan (1 leads to 6)mannose as the acceptor, the product was alphaMan(1 leads to 6)mannose and with alphaMan-(1 leads to 2)mannose the product was tentatively characterized as a mixture of alphaMan-(1 leads to 3)alphaMan(1 leads to 2)mannose and alphaMan(1 leads to 2)alphaMan(1 leads to 2)mannose. 4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation. 5. Radioactive disaccharides were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysaccharide to a minor extent under the same conditions.  相似文献   

2.
1. A microsomal enzyme preparation from the yeast Saccharomyces cerevisiae catalyzes the transfer of mannosyl units from GDPmannose to mannose and a number of mannose-containing oligosaccharides and glycosides whereby different glycosidic bonds are formed.2. Of the compounds tested besides mannose, only those containing an α-linked mannosyl unit at the nonreducing position of their moleculae were effective as receptors. Monodeoxyanalogues of mannose as well as α-mannose phosphates did not serve as receptors in the above reaction.3. The structure of the product formed with mannose as receptor was determined to be O-α-D-mannosyl-(1→2)-mannose; with αMan(1→Man(1→6)mannose as the acceptor, the product was αMan(1→6)αMan(1→6)mannose and with αMan-(1→2)mannose the product was tentatively characterized as a mixture of αMan-(1→3)αMan(1→2)mannose and αMan(1→2)αMan(1→2)mannose.4. The enzymes catalyzing the formation of different types of glycosidic bonds differed in their acceptor specificity, pH-activity curves and rates of heat denaturation.5. Radioactive disaccharids were unable to enter the mannan protein molecule in the cell-free system while free radioactive mannose did incorporate into polysacchride to a minor extent under the same conditions.  相似文献   

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An overview of mannan structure and mannan-degrading enzyme systems   总被引:2,自引:0,他引:2  
Hemicellulose is a complex group of heterogeneous polymers and represents one of the major sources of renewable organic matter. Mannan is one of the major constituent groups of hemicellulose in the wall of higher plants. It comprises linear or branched polymers derived from sugars such as d-mannose, d-galactose, and d-glucose. The principal component of softwood hemicellulose is glucomannan. Structural studies revealed that the galactosyl side chain hydrogen interacts to the mannan backbone intramolecularly and provides structural stability. Differences in the distribution of d-galactosyl units along the mannan structure are found in galactomannans from different sources. Acetyl groups were identified and distributed irregularly in glucomannan. Some of the mannosyl units of galactoglucomannan are partially substituted by O-acetyl groups. Some unusual structures are found in the mannan family from seaweed, showing a complex system of sulfated structure. Endohydrolases and exohydrolases are involved in the breakdown of the mannan backbone to oligosaccharides or fermentable sugars. The main-chain mannan-degrading enzymes include β-mannanase, β-glucosidase, and β-mannosidase. Additional enzymes such as acetyl mannan esterase and α-galactosidase are required to remove side-chain substituents that are attached at various points on mannan, creating more sites for subsequent enzymatic hydrolysis. Mannan-degrading enzymes have found applications in the pharmaceutical, food, feed, and pulp and paper industries. This review reports the structure of mannans and some biochemical properties and applications of mannan-degrading enzymes.  相似文献   

6.
One side chain in the cell wall mannan of the yeast Kluyveromyces lactis has the structure (see article). (Raschke, W. C., and Ballou, C. E. (1972) Biochemistry 11, 3807). This (Man)4GNAc unit (the N-acetyl-D-glucosamine derivative of mannotetroase) and the (Man)4 side chain, aMan(1 yields 3)aMan(1 yields 2)aMan(1 yields 2)Man, are the principle immunochemical determinants on the cell surface. Two classes of mutants were obtained which lack the N-acetyl-D-glucosamine-containing determinant. The mannan of one class, designated mmnl, lacks both the (Man)4GNAc and (Man)4 side chains. Apparently, it has a defective alpha-1 yields 3-mannosyltransferase and the (Man)4 unit must be formed to serve as the acceptor before the alpha-1 yields 2-N-acetyl-glucosamine transferase can act. The other mutant class, mnn2, lacks only the (Man)4GNAc determinant and must be defective in adding N-acetylglucosamine to the mannotetrasose side chains. Two members of this class were obtained, one which still showed a wild type N-acetylglucosamine transferase activity in cell-free extracts and the other lacking it. They are allelic or tightly linked, and were designated mnn2-1 mnn2-2. Protoplast particles from the wild type cells catalyzed a Mn2+-dependent transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the mannotetraose side chain of endogenous acceptors. Exogenous mannotetraose also served as an acceptor in a Mn2+-dependent reaction and yielded (Man)4GNAc. Related oligosaccharides with terminal alpha (1 yields 3)mannosyl units were also good acceptors. The product from the reaction with alphaMan(1 yields 3)Man had the N-acetylglucosamine attached to the mannose unit at the reducing end, which supports the conclusion that the cell-free glycosyltransferase activity is identical with that involved in mannan synthesis. The reaction was inhibited by uridine diphosphate. Protoplast particles from the mmnl mutants showed wild type N-acetylglucosamine transferase activity with exogenous acceptor, but they had no endogenous activity because the endogenous mannan lacked acceptor side chains. Particles from the mnn2-1 mutant failed to catalyze N-acetylglucosamine transfer. In contrast, particles from the mnn2-2 mutant were indistinguishable from wild type cells in their transferase activity. Some event accompanying cell breakage and assay of the mnn2-2 mutant allowed expression of a latent alpha-1 yields 2-N-acetylglucosamine transferase with kinetic properties similar to those of the wild type enzyme.  相似文献   

7.
A homogenate of mechanically broken, freshly grown Saccharomyces cerevisiae X2180 cells catalyzes the transfer of mannosylphosphate units from guanosine diphosphate mannose to reduced alpha1 leads to 2-[3H]mannotetraose to yield reduced mannosylphosphoryl [3H]-mannotetraose. The product is analogous in structure to the phosphorylated mannan side chains, which suggests that the enzymic activity is involved in mannoprotein biosynthesis in the intact cell. The mannosylphosphate transferase activity, localized in a membrane fraction obtained by differential centrifugation at 100,000 x g, was solubilized by Triton X-155 and purified 250-fold by ammonium sulfate precipitation and by ion exchange and gell filtration chromatographies. The enzyme requires MN2+ OR Co2+ ions for activity and is stimulated by various detergents. The mnn2 and mnn3 mannan mutants of S. cerevisiae possess normal levels of mannosylphosphate transferase activity, whereas the mnn4 mutant cells contain very low, if any, activity. This is consistent with a previous conclusion that the mnn4 mutation affects the mannosylphosphate transferase activity, whereas the mnn2 and mnn3 strains possess phosphate-deficient mannans because they are unable to synthesize the appropriate side chain precursors. A new mannan mutant class with the mnn4 chemotype was isolated, but the mutation proved to be recessive and nonallelic with the mnn4 locus. This new locus is designated mnn6.  相似文献   

8.
The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm1 and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect immunofluorescence microscopy and by transmission electron microscopy. The mdm1 lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm1 or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin- or actin-based cytoskeletons. The mdm1 and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.  相似文献   

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Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.  相似文献   

11.
Two yeast mutants defective in endocytosis are defective in pheromone response   总被引:43,自引:0,他引:43  
Y Chvatchko  I Howald  H Riezman 《Cell》1986,46(3):355-364
We have purified biosynthetically labeled alpha-factor secreted from transformed yeast alpha cells. This alpha-factor binds specifically to a cells and is internalized by a time-, temperature-, and energy-dependent process. alpha-factor is internalized in an intact form and then rapidly degraded. Two yeast mutants defective in the accumulation of an endocytotic marker, lucifer yellow CH, in the vacuole have been isolated. end1 accumulates invaginations of the plasma membrane, and end2, an internal membrane-bound organelle. One of these mutants, end1, is defective for internalization of alpha-factor. Both of these mutants are defective in pheromone response.  相似文献   

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R H Douglas  C E Ballou 《Biochemistry》1982,21(7):1561-1570
An enzyme activity in Kluyveromyces lactis that catalyzes the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine to alpha Man(1 leads to 3) alpha Man ( 1 leads to 2) alpha Man (1 leads to 2)Man to yield alpha Man(1 leads to 3) [alpha GlcNAc(1 leads to 2)] alpha Man(1 leads to 2) alpha Man (1 leads to 2)Man, a mannoprotein side-chain unit, has been solubilized by Triton X-100 and purified 18000-fold by a combination of ion-exchange chromatography, gel filtration, hydrophobic chromatography, and adsorption to a lectin column. The enzyme activity from a K. lactis mutant (mnn2-2) that made mannoprotein lacking N-acetylglucosamine in its side chains, but that possessed a normal level of transferase activity in cell extracts, was purified and compared with the enzyme from the wild-type strain. Both transferase activities are integral membrane proteins found in particles associated with endoplasmic reticulum. The two purified enzymes had the same apparent size, heat stability, Mn2+ requirement, and Km for donor and acceptor and a similar Vmax. Wild-type and mutant cells had similar pool sizes of sugar nucleotide donor, and they incorporated labeled N-acetylglucosamine into chitin at similar rates. No evidence was obtained for an inactive enzyme precursor in mutant cells that was activated upon breaking the cells, nor did the mutant cells contain a transferase inhibitor or a hexosaminidase that could remove the sugar from the mannoprotein during processing and secretion. The mnn2-2 locus appears to be allelic with a second mutant, mnn2-1, that has the same phenotype but that lacks transferase activity in cell extracts. This suggests that the two mutations affect the structural gene for the transferase, and we conclude that the mnn2-2 mutant could contain an altered enzyme that fails to function because it is improperly localized or oriented in the membrane.  相似文献   

14.
The Saccharomyces cerevisiae mutants affected in the structure of mannan outer chain were found to synthesize dolichol diphosphate-linked oligosaccharides identical in size to those of the wild type strain. The mannosyl transferases involved in the synthesis of the outer chain had an absolute requirement for manganese ions and were activated when enzymatic preparations were stored at 2 degrees C, whereas the transferases responsible for the formation of dolichol monophosphate mannose and dolichol diphosphate oligosaccharides were drastically inactivated from the onset of storage and required magnesium or manganese ions, the former being more effective than the latter. Both sets of enzymes could be separated by ion exchange chromatography. In vitro conditions that enhanced the synthesis of dolichol monophosphate mannose did not stimulate the incorporation of mannose residues into the outer chain. It is concluded that dolichol monophosphate mannose is not an intermediate in the synthesis of the outer chain and that this part of mannan and the dolichol diphosphate oligosaccharides are synthesized by different mannosyltransferases.  相似文献   

15.
The formation of two distinct types of carbohydrate moieties, β-elminable saccharides attached to serine and/or threonine and of the polysaccharide portion of yeast mannan-protein was investigated using the particulated mannan synthetase from Saccharomyces cerevisiae and GDP-[U-14C] mannose as mannosyl donor.The accumulated evidence obtained by following the kinetics of mannose incorporation into the different carbohydrate portions of mannoproteins, kinetics of thermal denaturation of enzymes responsible for the synthesis and by “pulse-chase” experiment where the change in the distribution of incorporated radioactivity was followed between the two carbohydrate moieties strongly suggests that the two carbohydrate portions in yeast mannoprotein are being synthesized independently, most probably by different sets of enzymes. At the same time, the obtained data show the β-eliminable saccharides attached to serine and threonine in the peptide do not serve as precursors in the formation of polysaccharide part of mannoprotein.  相似文献   

16.
《Autophagy》2013,9(2):278-280
Mitochondria autophagy (mitophagy) is the process of selective degradation of mitochondria that has an important role in mitochondrial quality control. To gain insight into the molecular mechanism of mitophagy, we screened a yeast knockout library for strains that are defective in mitophagy. We found 32 strains that showed a complete or partial block of mitophagy. One of the genes identified, YLR356W, is required for mitophagy, but not for macroautophagy or other types of selective autophagy. The deletion of YLR356W partially inhibits mitophagy during starvation, whereas there is almost complete inhibition at post-log phase. Accordingly, we hypothesize that Ylr356w is required to detect or present aged or dysfunctional mitochondria when cells reach the post-log phase.  相似文献   

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H Riezman 《Cell》1985,40(4):1001-1009
Yeast cells have been shown to internalize lucifer yellow CH by endocytosis. Internalization of the fluorescent dye is time-, temperature-, and energy-dependent, it is not saturable, and the dye is accumulated in the vacuole. Some of the yeast secretory mutants that accumulate endoplasmic reticulum or Golgi bodies are defective for endocytosis at restrictive temperature, while others are not. All of the mutants that accumulate secretory vesicles are defective for endocytosis. These results suggest that efficient transport of proteins from the endoplasmic reticulum to the Golgi apparatus and from the Golgi to secretory vesicles is not necessary for endocytosis. In contrast, endocytosis may be obligatorily coupled with the latest steps of secretion.  相似文献   

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