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1.
MCF7 cells were cultured with steroids, labelled with (35S)-methionine and the secreted and intracellular proteins were examined by one and two dimensional gel electrophoresis. Estradiol (0.1 nM) increased the synthesis of some of the secreted proteins; the induction of a protein of molecular weight 46,000 daltons being the most dramatic. The 46,000 daltons secreted protein was heterogeneous with respect to molecular weight and isoelectric point. The antiestrogen Tamoxifen did not stimulate the synthesis of any of the estrogen induced proteins, but completely inhibited the induction by estradiol. The effect of estradiol on internal proteins was much more subtle; only 3 proteins out of about 250 were stimulated. The functions of these Proteins are unknown, however they appear to be good markers for studying the mechanism of action of estrogens and antiestrogens in breast cancer and might be related to the control of cell proliferation by estrogen. 相似文献
2.
Identification and characterization of cellular targets for tyrosine protein kinases 总被引:20,自引:0,他引:20
Protein kinases associated with the transforming proteins of a number of retroviruses are specific for tyrosine. Several proteins in cells transformed by these viruses are phosphorylated at tyrosine. We have now identified three unrelated abundant nonphosphorylated cellular proteins of 46,000, 39,000 and 28,000 daltons in chick embryo cells, which are the unphosphorylated forms of phosphotyrosine-containing proteins and thus are substrates for tyrosine protein kinases. By two-dimensional gel analysis, we have found that the 46,000-dalton protein exists in two unphosphorylated forms of which the more acidic is a minor species. This latter form is phosphorylated, chiefly at serine, in both normal and transformed cells, generating a yet more acidic species. In transformed but not normal cells, the major form is phosphorylated at tyrosine and serine, yielding a fourth isoelectric variant. The 46,000-dalton unphosphorylated protein has been partially purified, and antiserum to it recognizes all four isoelectric variants of the protein. The 39,000-dalton protein has two unphosphorylated forms of which the more acidic is a minor species. The major form is phosphorylated at tyrosine and serine in transformed cells only. The 39,000-dalton unphosphorylated protein has been partially purified, and antiserum raised to it recognizes all three isoelectric variants. The 28,000-dalton protein has a single phosphorylated form which contains serine in normal cells, but both serine and tyrosine in transformed cells. 相似文献
3.
Treatment of intact normal rat kidney fibroblasts, or of purified NRK plasma membranes, with trypsin or papain markedly enhances adenylate cyclase activity [ATP pyrophosphatelyase (cyclizing) EC 4.6.1.1]. Limited proteolysis (25 μg/ml trypsin for 7 min) of confluent cells grown with unheated calf serum significantly increases cyclase activity, whereas similar treatment of sparse cells causes only a marginal increase in cyclic AMP formation. To determine which membrane protein(s) is altered under conditions which result in proteolytic activation of adenylate cyclase, purified plasma membranes and intact normal rat kidney cells were subjected to limited proteolysis and membrane proteins analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Membranes prepared from intact confluent normal rat kidney cells exposed to mild trypsinization showed a decrease in proteins of 56,000, 46,000, 37,000, and 32,000 daltons. Trypsin treatment of intact, sparse cells does not activate the cyclase system and does not lead to modification of the 46,000-dalton membrane protein. Treatment of purified normal rat kidney plasma membranes results in the loss of numerous bands in the high molecular mass region (>150,000 daltons) as well as decreases membrane proteins of 56,000, 49,000, 46,000, and 23,000 daltons. Compared with trypsin, the proteolytic action of papain appears to be quite specific, causing a discernible decrease in only the 46,000-dalton protein. The correlation between modification of the 46,000-dalton membrane component and the activation of the cyclase system suggests that perhaps this protein is proteolytically modified to elicit activation of adenylate cyclase. 相似文献
4.
C L Geczy 《Journal of immunology (Baltimore, Md. : 1950)》1977,119(6):2107-2113
Mitogenic factor from BCG-sensitized cells stimulated with antigen (PPD) was found to have a m.w. between 20 and 25,000 daltons and an isoelectric point of about 7.5. The blastogenic activity of this factor was not affected by L-fucose or heating at 56 degrees C for up to 1 hr. Mitogenic factor obtained from supernatants of allogeneic cell mixtures (MLC-MF) on the other hand, had a m.w. at 15 to 18,000 daltons and an isoelectric point of 6.5. The blastogenic activity of MLC-MF was inhibited by 0.1 M L-fucose. The factor was stable at 56 degrees C for 1 hr. An antibody prepared against MLC-MF inhibited the MLC reaction as well as the activity of MLC-MF on non-committed cells. This antibody, however, did not affect the response of lymphocytes to PHA or PPD and had no suppressive effect on PPD-MF. The antibody was not cytotoxic and its suppressive activity in the MLC response could not be absorbed out by lymphoid cells indicating that it is probably directed against a lymphocyte activation product (MLC-MF) rather than membrane antigens. The chemical and immunologic differences exhibited by PPD-MF and MLC-MF indicate that these two lymphokines are distinct molecular entities. 相似文献
5.
A simple and rapid method for the purification of glutathione S-transferase is described. The physical and kinetic properties of purified enzyme are reported. The protein is constituted of two identical subunits with a total molecular weight of 46,000 daltons. The isoelectric focusing of crude cytosol or purified preparation gives a single peak of activity with a pI of 7.1. The kinetic analysis shows a relatively strict substrate specificity. Only 1-chloro-2,4-dinitrobenzene is conjugated to reduced glutathione at an appreciable rate. The peroxidase activity of the enzyme with respect to cumene hydroperoxide as substrate is negligible. Hemin and bilirubin are competitive inhibitors of catalytic activity. 相似文献
6.
Identification of tubulin isoforms in the plasmodium of Physarum polycephalum by in vitro microtubule assembly 总被引:3,自引:0,他引:3
The tubulins of the plasmodium of Physarum polycephalum have been identified by in vitro microtubule assembly from partially purified extracts of asynchronous microplasmodia and late G2 macroplasmodia. The plasmodial tubulin group comprised of 2 alpha tubulins (app. m.w. 51000 daltons) and 2 beta tubulins (app. m.w. 58000 daltons and 55000 daltons) and appeared to be identical with a group of polypeptides which are synthesized periodically in late G2. Two of the plasmodial tubulin subunits (one alpha and one beta) were identical to the Physarum amoebal tubulin alpha and beta subunits as characterised by 2D gel positions. 相似文献
7.
Possible involvement of a cell surface glycoprotein in the differentiation of skeletal myoblasts 总被引:5,自引:0,他引:5
From a highly myogenic permanent line of rat skel-myoblasts (L6), we have isolated two classes of single step concanavalin A-resistant mutants. The RI class is about 2-fold and RII about 5-fold more resistant than the parental cells to the lethal action of concanavalin A. In all of the mutants, both the morphological differentiation (i.e. fusion to form myotubes) and biochemical differentiation, measured by the appearance of creatine kinase and acetylcholine receptors, are absent. The biochemical lesion in the RI type of mutants is not known, but RII type of mutants is unable to catalyze transfer of mannose from GDP-mannose into a lipid-linked form. Concanavalin A binding to separated membrane proteins from RII type of mutants on polyacrylamide gels is reduced 80% compared to wild type cells. In the RI type of mutants, however, only one major band, approximately 46,000 daltons, does not bind concanavalin A to the same extent as the wild type cells. In somatic cell hybridizations, RI type of mutants complements the RII type. In the hybrids, fusion as well as creatine kinase and acetylcholine receptors reappear, although not to the same extent as in the wild type cells. The 46,000-dalton band also reappears in the complementing hybrids. Thus, this protein may play some crucial role in myogenesis. 相似文献
8.
S A Fellini G S Bennett Y Toyama H Holtzer 《Differentiation; research in biological diversity》1978,12(2):59-69
The 100 A filament subunit proteins of chick fibroblasts and gizzard smooth muscle were compared. These proteins are major cellular components in these cell types, constituting up to 98% of the cell's total protein. Co-electrophoresis of cytoskeletal fractions of fibroblasts and smooth muscle revealed that the subunit proteins differed in their molecular weights: 58,000 daltons in fibroblasts and 55,000 daltons in smooth muscle. Cytoskeletal fractions from other cell types were also examined: chondroblasts contained the 58,000 dalton subunit, and cytoskeletons of skeletal muscle and cardiac muscle contained both 55,000 and 58,000 dalton proteins. Chick skin and rat kangaroo Pt K2 cells had more complex subunit patterns which resemble prekeratin. The peptide patterns resulting from proteolytic digestion of the 58,000 dalton protein of fibroblasts, the 55,000 dalton proteins of smooth muscle and PT K2 cells, and chick brain tubulin differed from one another. Two-dimensional electrophoresis of reconstituted gizzard smooth muscle 100 A filaments showed the 55,000 dalton subunit to be composed of two major components, differing in their isoelectric points. Antibodies prepared against electrophoretically purified 55,000 dalton subunit protein reacted in immunodiffusion against the original smooth muscle antigen and cytoskeletal fractions from skeletal and cardiac muscle, but not from fibroblasts, brain, liver, or skin cells. A specific antigenic determinant common to subunit proteins in smooth, skeletal, and cardiac muscle, is therefore indicated. A previously described antibody against fibroblast subunit protein reacted weakly against smooth muscle filament protein in immunodiffusion revealing the presence of a common antigenic determinant between the two subunit proteins. These data demonstrate striking antigenic and primary structural differences in 100 A filament subunits from even such closely related cell types as fibroblasts on the one hand and muscle cells on the other. 相似文献
9.
Purification and characterization of actobindin, a new actin monomer-binding protein from Acanthamoeba castellanii 总被引:5,自引:0,他引:5
Actobindin is a new actin-binding protein isolated from Acanthamoeba castellanii. It is composed of two possibly identical polypeptide chains of approximately 13,000 daltons, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and with isoelectric points of 5.9. In the native state, actobindin appears to be a dimer of about 25,000 daltons by sedimentation equilibrium analysis. It contains no tryptophan and probably no tyrosine. Actobindin reduces the concentration of F-actin at steady state and inhibits the rate of filament elongation to extents consistent with the formation of a 1:1 actobindin-G-actin complex in a reaction with a KD of about 5 microM. The available data do not eliminate the possibility of other stoichiometries for the complex, but they are not consistent with any significant interaction between actobindin and F-actin. Despite the similarities between the effects of actobindin and Acanthamoeba profilin on the polymerization of Acanthamoeba actin, the two proteins are quite distinct with different native and subunit molecular weights, different isoelectric points, and different amino acid compositions. Also, unlike profilin, actobindin binds as well to rabbit skeletal muscle G-actin and to pyrenyl-labeled G-actin as it does to unmodified Acanthamoeba G-actin. 相似文献
10.
William A. Warren 《Preparative biochemistry & biotechnology》2013,43(3):199-208
Human skeletal muscle creatine kinase was purified by isoelectric focusing with a yield of greater than 60%. Two enzymic proteins, differing in specific activity, were obtained, and each final product produced only a single protein band when examined by electrophoretic methods. The proteins were composed of two subunits of about 41, 000 daltons each, and the amino acid compositions were similar. 相似文献
11.
The 46,000-dalton tyrosine protein kinase substrate is widespread, whereas the 36,000-dalton substrate is only expressed at high levels in certain rodent tissues 总被引:18,自引:7,他引:11
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《The Journal of cell biology》1984,98(2):487-497
Proteins of molecular mass 46,000 (p46) and 34,000-39,000 (p36) daltons are phosphorylated at tyrosine in Rous sarcoma virus-transformed chicken and mouse fibroblasts. p46 has recently been identified as an isozyme of enolase but the function of p36 is unknown. The expression of these proteins in various mouse and rat tissues has been examined. In most tissues, except muscle, p46 is found at relatively constant levels. In muscle, a more basic, related protein is present. In contrast, the abundance of p36 varies more widely from tissue to tissue, suggesting that it has a function in some but not all differentiated cells. By SDS gel electrophoresis and immunoblotting, high levels of p36 (60-120% of its relative abundance in fibroblasts) were found in small intestine, lung, and thymus, and intermediate levels (20-50%) were found in spleen, lymph nodes, and testes. No p36 was detectable in brain and muscle. Where studied, p36 mRNA expression paralleled protein levels. The cell types within each tissue expressing p36 were identified by immunofluorescence and immunoperoxidase staining. These cell types include all endothelial cells and fibroblastic cells examined, as well as various epithelial cells, cardiac muscle cells, macrophages, and testicular interstitial cells. We were unable to detect p36 in skeletal or smooth muscle cells, erythrocytes, nerve cells, or lymphocytes in any of the examined tissues. p36 appears to be concentrated in the terminal web region of intestinal columnar epithelial cells. 相似文献
12.
Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition 总被引:2,自引:0,他引:2
Antiserum against calf thymus dUTPase was raised in rats by injection of the partially purified enzyme. The antiserum did not react with dUTPase purified from rat spleen, while antibody against rat spleen dUTPase partially reacted with calf thymus enzyme. Native molecular weight of the calf thymus dUTPase was estimated at 46,000 daltons by gel filtration, and the denatured form of the enzyme was about 22,000, as judged by immunoblot analyses using the antibodies. These results indicate that the calf thymus dUTPase is composed of two identical subunits. 相似文献
13.
Discrete primary locations of a tyrosine-protein kinase and of three proteins that contain phosphotyrosine in virally transformed chick fibroblasts 总被引:25,自引:6,他引:19
We have studied the localization of three abundant cellular proteins which are substrates for tyrosine protein kinases in virally transformed chicken embryo fibroblasts. The primary location of each substrate is unaltered by transformation with Rous sarcoma virus (RSV). The tyrosine-phosphorylated species is localized with the nonphosphorylated species. Two of the proteins, of about 46,000 and 28,000 daltons, have a similar location. They are present in the high speed supernatant of cells homogenized in hypotonic buffer, and are soluble in nonionic detergent. The third protein, of about 39,000 daltons, is particulate when cells are homogenized in hypotonic buffer containing divalent cations, but approximately 30% is free in the high- speed supernatant when divalent cations are absent. This protein appears to be associated with the detergent-insoluble matrix when adherent cells are gently lysed in nonionic detergent in situ, but is soluble when the same cells are extracted with nonionic detergent in suspension. This suggests that one of the proteins are tightly associated with detergent-insoluble cytoskeletal structures, unlike the RSV transforming protein itself, which is the main tyrosine protein kinase known to be active in RSV-transformed cells. 相似文献
14.
Experiments were conducted to identify antigens of Trypanosoma cruzi (Brazil strain) to which antibodies are directed during the course of experimental Chagas' disease in C3H(He) (susceptible) and C57BL/6J (resistant) female mice. An extract of culture forms of the parasite was subjected to SDS-polyacrylamide gel electrophoresis, transferred to a solid phase matrix of nitrocellulose and used as antigens to detect antibodies in the sera of infected mice. Reactive antibodies were detected using an avidin-biotin peroxidase test. Two antigens were consistently detected with sera of normal, uninfected C57BL/6 and C3H(He) mice (51,000 and 44,000; and 53,000 and 46,000 daltons, respectively). A total of 32 antigens with m.w. of 230,000 to 25,000 daltons reacted with antibodies in sera of C3H mice infected for 25 days. Both the number of antigens detected and intensity of reactions increased with time of infection in C3H mice. An early (day 5), rapid, although weak response was observed to antigens of 85,000, 56,000, 53,000, 46,000 and 41,000 daltons. Throughout infection intense responses to antigens of 75,000, 67,000, 45,000, 41,000 and 36,000 daltons were detected. A similar number of components (a total of 34) with m.w. of 210,000 to 20,000 daltons were detected as being antigenic during the course of T. cruzi infection of C57BL/6 mice. A high number of antigens (25) was observed early in infection of C57BL/6 mice by day 10, including components with m.w. of 90,000, 85,000 and 70,000 daltons. Only minor changes were detected, however, after day 20 until day 120, when increases in the number of antigens and the intensity of certain reactions (e.g., antigens of 75,000, 46,000 and 26,000 daltons) were detected.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
15.
Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line, RPMI 4265. 总被引:1,自引:0,他引:1
M J Turner P Cresswell P Parham J L Strominger D L Mann A R Sanderson 《The Journal of biological chemistry》1975,250(12):4512-4519
HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed. 相似文献
16.
《Experimental mycology》1982,6(3):259-267
Sclerotia of Sclerotinia sclerotiorum contained a major protein of about 36, 000 daltons which was not detected in vegetative cells. The protein accumulated rapidly during sclerotial formation and ultimately comprised about 35–40% of the mature sclerotial protein. The protein did not decrease in concentration in sclerotial residue or appear in vegetative cells when sclerotia germinated myceliogenically. In contrast, the concentration of the protein decreased in sclerotia undergoing carpogenic germination and a small amount of the protein was present in the resultant apothecia. This development-specific protein was purified to near homogeneity as judged by one-dimensional polyacrylamide gel analysis. However, at least two other minor protein bands were detected by two-dimensional gel analysis which may be modified forms of the major protein. The protein had an isoelectric point of 6.0 and contained all 20 amino acids commonly present in proteins. 相似文献
17.
The IL 2 receptor isolated from the IL 2-dependent CTL-L cell line was subjected to biochemical analysis. Pulse-chase and tunicamycin studies, as well as digestion with the endoglycosidases, Endo-F and Endo-H, of 35S-methionine-labeled IL 2 receptors suggested a single protein precursor of 32,000 (p32) daltons. The p32 precursor was rapidly processed by addition of high-mannose-containing core N-linked sugars to intracytoplasmic precursor intermediates of 38,000 (p38) and 40,000 (p40) daltons, which undergo further processing to yield a mature surface receptor with heterogeneous apparent m.w. of 52,000 to 65,000 (p58). Two-dimensional gel studies indicated that p58 exhibited broad charge heterogeneity between pH 4.6 and 6.3. Endo-F digestions of p58 shifted the isoelectric focus point to a more basic 5.5 to 7.4. This considerable charge heterogeneity is consistent with the possibility that other posttranslational modifications to the mouse IL 2 receptor occur besides addition of complex N-linked glycans. Immunoprecipitations of the IL 2 receptor from surface iodinated cells also revealed an additional band at 110,000 (p110) daltons. IEF vs SDS-PAGE two-dimensional gel studies demonstrated that p110 also had an isoelectric focus point identical to p58. Western blot studies with an anti-IL 2 receptor monoclonal antibody (7D4) demonstrated that p38, p40, p58, and p110 each expressed the epitope recognized by this antibody. Thus, it is likely that p110 is not a unique molecule that coprecipitates with the IL 2 receptor. Western blot analysis of mitogen-stimulated T and B lymphocytes also revealed bands similar to p58 and p110, although these bands had an average apparent m.w. 3000 to 6000 less than those seen for CTL-L cells. 相似文献
18.
Purification of the Chick Eye Ciliary Neuronotrophic Factor 总被引:26,自引:11,他引:26
Dissociated 8-day chick embryo ciliary ganglionic neurons will not survive for even 24 h in culture without the addition of specific supplements. One such supplement is a protein termed the ciliary neuronotrophic factor (CNTF) which is present at very high concentrations within intraocular tissues that contain the same muscle cells innervated by ciliary ganglionic neurons in vivo. We describe here the purification of chick eye CNTF by a 2 1/2-day procedure involving the processing of intraocular tissue extract sequentially through DE52 ion-exchange chromatography, membrane ultrafiltration-concentration, sucrose density gradient ultracentrifugation, and preparative sodium dodecyl sulfate-polyacrylamide gradient electrophoresis. An aqueous extract of the tissue from 300 eyes will yield about 10-20 micrograms of biologically active, electrophoretically pure CNTF with a specific activity of 7.5 X 10(6) trophic units/mg protein. Purified CNTF has an Mr of 20,400 daltons and an isoelectric point of about 5, as determined by analytical gel electrophoresis. In addition to supporting the survival of ciliary ganglion neurons, purified CNTF also supports the 24-h survival of cultured neurons from certain chick and rodent sensory and sympathetic ganglia. CNTF differs from mouse submaxillary nerve growth factor (NGF) in molecular weight, isoelectric point, inability to be inactivated by antibodies to NGF, ability to support the in vitro survival of the ciliary ganglion neurons, and inability to support that of 8-day chick embryo dorsal root ganglionic neurons. Thus, CNTF represents the first purified neuronotrophic factor which addresses parasympathetic cholinergic neurons. 相似文献
19.
Characterization of superoxide dismutases purified from either anaerobically maintained or aerated Bacteroides gingivalis. 总被引:4,自引:2,他引:2
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A Amano S Shizukuishi H Tamagawa K Iwakura S Tsunasawa A Tsunemitsu 《Journal of bacteriology》1990,172(3):1457-1463
Superoxide dismutases (SODs) were purified from extracts of either anaerobically maintained or aerated Bacteroides gingivalis. Each purified enzyme (molecular weight, 46,000) was a dimer composed of two subunits of equal sizes. SOD from anaerobically maintained cells (anaero-SOD) contained 1.79 g-atom of Fe and 0.28 g-atom of Mn, and SOD from aerated cells (aero-SOD) contained 1.08 g-atom of Mn and 0.36 g-atom of Fe. Spectral analysis showed that anaero-SOD had the characteristic of Fe-SOD and that aero-SOD had that of Mn-SOD. Both enzyme preparations contained three isozymes with identical isoelectric points. On the basis of inactivation of SOD by H2O2, it was found that aero-SOD consisted of one Mn-SOD and a small quantity of two Fe-SODs, whereas anaero-SOD contained only Fe-SOD. However, each apoprotein from anaero-SOD and aero-SOD, prepared by dialysis in guanidinium chloride plus 8-hydroxyquinoline, showed only one protein band each with the same isoelectric point on an isoelectric focusing gel. Subsequent dialysis of both apoenzymes with either MnCl2 or Fe(NH4)2(SO4)2 restored the activity. These reconstituted SODs showed only one protein band with SOD activity on native polyacrylamide gel electrophoresis. Furthermore, the two enzymes had similar amino acid compositions, and their amino-terminal sequences were identical through the first 12 amino acids. These results suggest that the three isozymes of anaero-SOD and aero-SOD in B. gingivalis are formed from a single apoprotein. 相似文献
20.
Penicillium oxalicum Curie et Thom produced an exo-polygalacturonase, an endo-polygalacturonase and an endo-pectate lyase. It also produced cellulase Cx, cellobiase, B-glucosidase, xylanase, galactanase and arabinanase in culture and in infected yam tissues.The exo-polygalacturonase was unstable. It has a molecular weight of about 38 000 daltons. The endopolygalacturonase and endo-pectate lyase had an optimal pH of 5.0 and 8.5 and an isoelectric point (pI) at pH 3.6 and 4.9 respectively. Molecular weights of both enzymes were about 28 500 and 30 000 daltons respectively. The end products of the reaction were oligogalacturonides and revealed that cellulose was degraded to glucose, with cellobiose as an intermediate product. The end products of the degradation of hemicelluloses were xylose, galactose and arabinose. 相似文献