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1.
We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of -(14)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls transferred [14C]Gal from UDP-[14C]Gal into intact and partially hydrolyzed lupin -(14)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20–25°C in the presence of 25 mM Mn2+ and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan (M
r>150,000) from lupin we used was very low but increased when the M
r of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high-M
r (average M
r 60,000) -(14)-galactan was a more efficient acceptor [specific activity 2,000–3,000 pmol min–1 (mg protein)–1] than low-M
r (average M
r 10,000 and 5,000) polymers. Digestion of the radiolabeled product from high-M
r galactan with endo--(14)-galactanase released mainly radioactive -(14)-galactobiose and Gal, indicating that the transfer of [14C]Gal occurred through -(14)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) -(14)-galactooligomers with degree of polymerization at least 5. Gal7-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal8–10-PA.Abbreviations
AGP
Arabinogalactan-protein
-
Ara
Arabinose
-
DP
Degree of polymerization
-
GalA
Galacturonic acid
-
Gal
n
-PA
Pyridylaminated -(14)-galactooligosaccharides
-
GalT
Galactosyltransferase
-
MALDI–TOF–MS
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
-
Rha
Rhamnose
Sugars described in this paper belong to the d-series unless otherwise noted 相似文献
2.
Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM
r=52,000. During a 1.5-hr pulse-label with35S-methionine,-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM
r=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM
r=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M
r=62,000), with the remainder retained intracellularly (M
r=60,000). In the other two fucosidosis cell lines,-L-fucosidase was synthesized as an intracellular form withM
r=56,000 that was processed to an extracellular form withM
r=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby. 相似文献
3.
M. O. Ilori O. O. Amund O. Omidiji 《World journal of microbiology & biotechnology》1995,11(5):595-596
An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K
m value for starch was 0.27 mg ml-1 and its M
r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca2+, Mg2+, Na+ or K+ and inhibited by EDTA, KCN, citric acid and l-cysteine. 相似文献
4.
Yoshiharu Matahira Atsushi Tashiro Toshinari Sato Hirokazu Kawagishi Taichi Usui 《Glycoconjugate journal》1995,12(5):664-671
N-acetylhexosaminidase fromNocardia orientalis catalysed the synthesis of lacto-N-triose II glycoside (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OMe,3) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OMe (4) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OMe (5) throughN-acetylglucosaminyl transfer fromN,N-diacetylchitobiose (GlcNAc2) to methyl -lactoside. The enzyme formed the mixture of trisac-charides3, 4 and5 in 17% overall yield based on GlcNAc2, in a ratio of 20:21:59. Withp-nitrophenyl -lactoside as an acceptor, the enzyme also producedp-nitrophenyl -lacto-N-trioside II (-d-GlcNAc-(1-3)--d-Gal-(1-4)--d-Glc-OC6H4NO2-p,6) with its isomers -d-GlcNAc-(1-6)--d-Gal-(1-4)--d-Glc-OC6H4NO2-p (7) and -d-Gal-(1-4)-[-d-GlcNAc-(1-6)]--d-Glc-OC6H4NO2-p (8). In this case, when an inclusion complex ofp-nitrophenyl lactoside acceptor with -cyclodextrin was used, the regioselectivity of glycosidase-catalysed formation of trisaccharide glycoside was substantially changed. It resulted not only in a significant increase of the overall yield of transfer products, but also in the proportion of the desired compound6.Abbreviations GlcNAc2
2-acetamido-2-deoxy--d-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-d-glucose
- NAHase
N-acetylhexosaminidase
- -CD
-cyclodextrin 相似文献
5.
Henning Juhl 《Molecular and cellular biochemistry》1979,26(1):19-27
Summary cAMP independent glycogen synthase kinase and phosvitin kinase activity was purified from the 180 000 × g supernatant of human polymorphonuclear leukocytes by ammonium sulphate precipitation and phosphocellulose chromatography. The cAMP independent glycogen synthase kinase eluted from the phosphocellulose at 0.54 m NaCl (peak A) separate from the major phosvitin kinase eluting at 0.68 m NaCl (peak B). The kinase activity of both peaks tended to form aggregates, but in the presence of 0.6 m NaCl, the peak B enzyme had Mr 250 000, 7.2S and the peak A enzyme Mr 38 000, 3.8S. The ratio between synthase kinase and phosvitin kinase activity in peak A was 1:3.2 and in peak B 1:31.4. In addition the kinase activities differed with respect to sensitivity to temperature, ionic strength and CaCl2. It is suggested that the peak A enzyme represents the cAMP independent glycogen synthase kinase of leukocytes, whereas the peak B enzyme is a phosvitin kinase, which is insignificantly contaminated with some synthase kinase (peak A) and contains a separate, second synthase kinase.Synthase kinase had K
m
app
4.2 m for muscle glycogen synthease I and K
m
app
45 m for ATP. GTP was a poor substrate. The activity was not influenced by cyclic nucleotides, Ca2+, or glucose-6-P. Synthase I from muscle and leukocytes was phosphorylated to a ratio of independence of less than 0.05.Abbreviations cAMP
adenosine cyclic 3:5-monophosphate
- DTT
dithiothreitol
- EGTA
ethylene glycol-bis-(-amino-ethylether)-N,N-tetraacetic acid
- PMSF
phenylmethylsulfonylfluoride
- PKI
protein kinase inhibitor
- RI
ratio of independence for glycogen synthase
- SDS
sodium dodecyl sulphate 相似文献
6.
Serenella Eppenberger-Castori Hansruedi Lötscher Jukka Finne 《Glycoconjugate journal》1989,6(1):101-114
TheN-acetylglucosaminide (1–3/4)fucosyltransferase has been purified 1.8×106-fold from human milk by ion-exchange chromatography, affinity chromatography of GDP-agarose and HPLC. The (1–3/4)fucosyltransferase behaves in gel filtration-HPLC as a molecule of Mr 98 000, and differs from the (1–3)fucosyltransferase which behaves like a molecule of about Mr 47 000. The enzyme is a glycoprotein, and the purified preparation appears in SDS polyacrylamide gel electrophoresis as a band of Mr 44 000. The results present the first purification of human milk (1–3/4)fucosyltransferase to apparent homogeneity, and suggest that the (1–3/4)- and (1–3)fucosyltransferases of human milk differ in their native molecular sizes, the former being a dimer of two subunits. 相似文献
7.
Particulate membrane preparations isolated from cambial cells and differentiating and differentiated xylem cells of pine (Pinus sylvestris L.) trees synthesised [14C]glucans using either guanosine 5-diphosphate (GDP)-D-[U-14C]glucose or uridine 5-diphosphate (UDP)-D-[U-14C]glucose as glycosyl donors. Although these glucans had -(13) and -(14) linkages in an approximate ratio 1:1, the distribution of the linkages in the glucan synthesised from GDP-D-glucose was different from that synthesised from UDP-D-glucose. The synthesis of the mixed -(13) and -(14) glucan from GDP-D-[U-14C]glucose was changed to that of -(14) glucomannan in the presence of increasing concentrations of GDP-D-mannose. The glucan formed from UDP-D-[U-14C]glucose was not affected by any concentration of GDP-D-mannose. The membrane preparations epimerized GDP-D-glucose to GDP-D-mannose; however, the low amount of GDP-D-mannose formed was not incorporated into the polymer becaus the affinity of the synthase for GDP-D-glucose was much greater than that for GDP-D-mannose. The glucan formed from GDP-D-glucose and the glucomannan formed from GDP-D-glucose together with GDP-D-mannose were characterized. The apparent K
m and V
max of the glucan synthase for GDP-D-glucose were 6.38 M and 5.08 M·min-1, respectively. No lipid intermediates were detected during the synthesis of either glucan or glucomannan. The results indicated that an enzyme complex for the formation of the glucomannan was bound to the membrane.Abbreviations GDP
guanosine 5-diphosphate
- GLC
gasliquid chromatography
- UDP
trridine 5-diphosphate 相似文献
8.
Summary Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 × g supernatant, followed by DEAF-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had Mr 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had Mr 33 000 upon gelfiltration, but sedimented at 2.8–3.0S and 3.0–3.2S, respectively. R2 and R4 subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)–5.1S, respectively. The holoenzyme from peak I had Mr 165 000, 6.7S, which suggest a R2C2 structure, while that of peak III sedimented at 6.7S, but eluted at Mr 330 000 (2R2C2) by gelfiltration.The K
m
app
for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 m and 23 m, and cAMP 5 × 10–8
m and 6.3 × 10–8
m. Both enzymes had pH optimum 6.7–6.9 and were equally sensitive to Ca2+ temperature and protein kinase inhibitor. The substrate specificity was histone VS histone IIA = histone VIS casein > phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20–30% of cAMP dependent protein kinase activity and is absent from the 180 000 × g supernatant of gently disrupted cells.Purified catalytic subunit had K
m
app
(ATP) 20 m with rabbit muscle glycogen synthase I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07).cAMP independent histone kinase activity eluted in one peak (Peak II) at 3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with Mr 78 000. K
m
app
for ATP was 78 m and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca2+, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA > histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.Abbreviations AR
activity ratio for cAMP dependent protein kinase
- cAMP
adenosine cyclic 3:5-monophosphate
- cIMP
inosine cyclic 3:5-monophosphate
- cGMP
guanosine cyclic 3:5-monophosphate
- Glucose-6-P
glucose-6-phosphate
- DDT
dithiothreitol
- EGTA
ethylene glycol-bis-(-aminoethylether)-N, N-tetraacetic acid
- PMSF
phenylmethylsulfonylfluoride
- PKI
protein kinase inhibitor
- RI
ratio of independence for glycogen synthase
- SDS
sodium dodecyl sulphate 相似文献
9.
Three allophycocyanin complexes were separated by gel electrophoresis, isoelectric focusing and ion exchange chromatography from a low molecular fraction (Mr 100–150000) of partially dissociated phycobilisomes of Mastigocladus laminosus: A. (APAP); B. (*AP2
AP2
AP*AP) · L
C
10
; and C. (*APAPBAP2
AP*AP) · L
C
10
. According to their fluorescence emission maximum at room temperature the complexes A., B. and C. are designated AP 660, AP 664 and AP 680. The different subunits of the AP complexes have apparent molecular weights of Mr 18500 *AP, 18200 APB, 18000 AP, 17000 AP and 16500 *AP. This hitherto unrecognized microheterogeneity within the AP subunits of complexes B. and C. of Mastigocladus laminosus phycobilisomes could also be demonstrated and confirmed with the two phycocyanin complexes PC 642 and PC 646. PC 642 is characterized by a L
R
11
linker polypeptide.Abbreviations AP
allophycocyanin
- PC
phycocyanin
- PEC
phycoerythrocyanin
- PE
phycoerythrin
- PAGE
polyacrylamide gel electrophoresis
- IEF
isoelectric focusing
- pI
isoelectric point
- Mr
apparent molecular weight
- TMED
tetramethylethylenediamine
- APS
ammonium persulphate
- SDS
sodium dodecylsulphate
- O.D.
optical density
A preliminary account of this work has been presented at the Embo Workshop on Oxygenic and Anoxygenic Electron Transport Systems in Cyanobacteria (Blue-green Algae) in Cape Sounion, Greece, 20–25 September 1987 相似文献
10.
Heidrun Lorenzl Fred Bernd Oppermann Bernhard Schmidt Alexander Steinbüchel 《Antonie van Leeuwenhoek》1993,64(1):9-15
InClostridium magnum strain Wo Bd P1 the formation of the enzyme components of the acetoin dehydrogenase enzyme system E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase Ao:DCPIP OR), E2 (dihydrolipoamide acetyltransferase DHLTA) and E3 (dihydrolipoamide dehydrogenase DHLDH) were induced during growth on acetoin. Ao:DCPIP OR was purified from acetoin-grown cells in two steps by chromatography on DEAE-Sephacel and on Mono Q HR. Native Ao:DCPIP OR exhibited a Mr of 138,000; it consisted of two different subunits of Mr 38,500 and Mr 34,000, and it occurred most probably in a tetrameric 22 structure. The N-terminal amino acid sequences of the - and -subunits revealed homologies to the N-termini of the corresponding subunits of Ao:DCPIP OR fromPelobacter carbinolicus and fromAlcaligenes eutrophus; furthermore, the N-terminus of the -subunit exhibited homologies to the N-termini of -subunits from different 2-oxo acid dehydrogenases.Abbreviations Ao:DCPIP OR
acetoin:2,6-dichlorophenolindophenol oxidoreductase
- DHLDH
dihydrolipoamide dehydrogenase
- DHLTA
dihydrolipoamide acetyltransferase
- HETPP
hydroxyethyl thiamine pyrophosphate 相似文献
11.
Jean-Michel Wieruszeski Jean-Claude Michalski Jean Montreuil Gérard Strecker 《Glycoconjugate journal》1990,7(1):13-26
The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Leb and B Leb healthy subjects, were each found to be contaminated by a minor component when analysed by1H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and1H/13C-NMR spectroscopy.Abbreviations HPLC
high performance liquid chromatography
- GLC
gas liquid chromatography
- NMR
nuclear magnetic resonance
- COSY
correlation spectroscopy
- Gal
d-galactopyranose
- GalNAc
2-acetamido-2-deoxy-d-galactopyranose
- Glc
d-glucopyranose
- Fuc
l-fucopyranose
- LNDFH I
lacto-N-difucohexaose I (Leb determinant 相似文献
12.
The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [3H]glucosamine and [35S]Na2SO4 by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS
3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate
- Di-OS
2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose
- Di-4S
2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose
- Di-6S
2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose
- Gdn-HCl
guanidine hydrochloride
- WGA
wheat germ agglutinin 相似文献
13.
Summary A vanadate-sensitive H+-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H+-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H+ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H+ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na3VO4) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK
i
for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na3VO4). ATPase activity of the reconstituted vesicles showed aK
i
of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme. 相似文献
14.
The free-living cyanobacterium Anabaena variabilis showed a biphasic pattern of 14CH3NH
3
+
uptake. Initial accumulation (up to 60 s) was independent of CH3NH
3
+
metabolism, but long-term uptake was dependent on its metabolism via glutamine synthetase (GS). The CH3NH
3
+
was converted into methylglutamine which was not further metabolised. The addition of l-methionine-dl-sulphoximine (MSX), to inhibit GS, inhibited CH3NH
3
+
metabolism, but did not affect the CH3NH
3
+
transport system.NH
4
+
, when added after the addition of 14CH3NH
3
+
, caused the efflux of free CH3NH
3
+
; when added before 14CH3NH
3
+
, NH
4
+
inhibited its uptake indicating that both NH
4
+
and CH3NH
3
+
share a common transport system. Carbonylcyanide m-chlorophenylhydrazone and triphenyl-methylphosphonium both inhibited CH3NH
3
+
accumulation indicating that the transport system was -dependent. At pH 7 and at an external CH3NH
3
+
concentration of 30 mol dm-3, A. variabilis showed a 40-fold intracellular accumulation of CH3NH
3
+
(internal concentration 1.4 mmol dm-3). Packets of the symbiotic cyanobacterium Anabaena azollae, directly isolated from the water fern Azolla caroliniana, also showed a -dependent NH
4
+
transport system suggesting that the reduced inhibitory effect of NH
4
+
on nitrogenase cannot be attributed to the absence of an NH
4
+
transport system but is probably related to the reduced GS activity of the cyanobiont.Abbreviations CCCP
carbonylcyanide m-chlorophenylhydrazone
- GS
glutamine synthetase
- HEPES
4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid
- MSX
l-methionine-dl-sulphoximine
-
membrane potential
- pH
transmembrane pH difference
- TPMP+
triphenylmethylphosphonium 相似文献
15.
Filamentous fungi are capable of secreting relatively large amounts of heterologous recombinant proteins. Recombinant human glycoproteins expressed in this system, however, carry only carbohydrates of the oligomannose type limiting their potential use in humans. One approach to the problem is genetic engineering of the fungal host to permit production of complex and hybrid N-glycans. UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (GnT I) is essential for the conversion of oligomannose to hybrid and complex N-glycans in higher eukaryotic cells. Since GnT I is not produced by fungi, we have introduced into the genome ofAspergillus nidulans the gene encoding full-length rabbit GnT I and demonstrated the expression of GnT I enzyme activity at levels appreciably higher than occurs in most mammalian tissues. All the GnT I activity in theAspergillus transformants remains intracellular suggesting that the rabbit trans-membrane sequence may be capable of targeting GnT I to the fungal Golgi apparatus.Abbreviations CM
complete medium
- Gal-T
UDP-Gal:GlcNAc -1,4-galactosyltransferase (EC 2.4.1.38/90)
- GnT I
UDP-GlcNAc:3-d-mannoside -1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101)
- HPLC
high performance liquid chromatography
- M3-octyl
Man1-6[Man1-3]Man-octyl
- PAGE
polyacrylamide gel electrophoresis
- MES
2-(N-morpholino)ethane sulfonate
- PCR
polymerase chain reaction
- PEG
polyethylene glycol
- PMSF
phenyl methyl sulfonyl fluoride
- SDS
sodium dodecyl sulfate
- SSC (1×)
0.15m NaCl/0.015m sodium citrate (pH 7.0)
- STC
1.2m sorbitol, 100mm Tris-HCl, pH 7.4, and 10mm CaCl2
- STET
0.1m NaCl, 10mm Tris-HCl, pH 8.0, 1mm EDTA, pH 8.0, 5% Triton-X-100
Deceased. This paper is dedicated to the memory of Lorne S. Reid. 相似文献
16.
-Galactosidase II2 (MW 43 390) from resting Vicia faba L. seeds had been shown to possess d-glucose/d-mannose-specific lectin activity. Inhibition studies with monosaccharides and an examination of the effects of heat and pH on the catalytic and lectin activities of the enzyme indicate that the enzyme substrate and the lectin haptens bind at different sites on the protein. d-Mannosebinding has been investigated by equilibrium dialysis and spectrophotometrically. Both methods yield Ka values of approx. 3·103 M-1 for the interaction and there would appear to be two mannosebinding sites per molecule of enzyme protein. The lectin properties of V. faba -galactosidase II2 have been discussed in relation to both V. faba lectin (favin) and other legume -galactosidases.Abbreviations con A
concanavalin A
- CM-cellulose
carboxymethyl cellulose
- MW
molecular weight
- PNPG
p-nitrophenyl -d-galactoside
- SDS
sodium dodecyl sulphate
- PAGE
polyacrylamide-gel electrophoresis 相似文献
17.
The aluminium (Al) tolerance of 34 temperate legume species (143 genotypes, including 57 from Trifolium repens) was determined in 60 experiments over a 3 year period in a low ionic strength (2.7 × 10-3
M) solution culture. For each genotype, the relationship between solution Al3+ activity (M) and relative yield was determined and the Al3+ activity associated with a 50% reduction in yield (AlRY50) calculated. In addition, plant chemical concentrations were determined in at least one genotype from most species. For white clover, AlRY50 over all genotypes had an approximately normal distribution with mean of 1.31 M for the tops and 1.51 M for the roots, and a standard deviation of about 0.4. This suggested that Al tolerance had a polygenic inheritance. For the other species tested, AlRY50 ranged from 0.15 to 4.53 M in the tops and from 0.21 to 4.89 M in the roots. In the tops and roots, 37% and 26% respectively of the genotypes had an AlRY50 less than 1 M, including all species tested in the genera Melilotus and Medicago. Only 8% or 23% of the genotypes, based on the tops and roots respectively, had an AlRY50 greater than 2, including all genotypes in the species Lotus pedunculatus. Except for Lotus, there were no consistent differences between genera in plant chemical concentrations. In Lotus, concentrations of Ca, Zn, Mn and Cu in the tops and of all elements except B in the roots were lower than that of the other species. The AlRY50 of the species was not related to plant chemical concentrations in the absence of Al. Depending on the plant element, increasing solution Al concentrations had no significant effect on plant chemical concentrations for 56–94% of the species. When a significant effect did occur, increasing Al in solution generally decreased S and K concentrations and increased Mn, Zn, Cu Fe, B and Al concentrations in the tops and roots and decreased Ca concentrations in the tops. Plant P concentrations decreased in the tops but increased in the roots. Increasing Al in solution increase plant Al at the average rate of 44 g g-1 M
-1 (range 20–87) in the tops and 333 g M
-1 (range 162–616) in the roots. 相似文献
18.
The quinol oxidase appears to be mainly responsible for the oxidation of bacterial MKH2 in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mol cytochrome aa
3 per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa
3 was about 103 electrons · s-1 at pH 7 and 37°C. The preparation consisted mainly of a M
r 57000 and a M
r 36000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M
r 36000 polypeptide.Abbreviations DMN
2,3-dimethyl-1,4-naphthoquinone
- DMNH2
2,3-dimethyl-1,4-naphthoquinol
- Duroquinol
2,3,5,6-tetramethyl-1,4-benzoquinol
- MK
menaquinone
- MKH2
menaquinol
- NBH2
2,3-dimethoxy-5-methyl-6-(n-nonyl)-1,4-benzoquinol
- TMPD
N,N,N, N,-tetramethyl-1,4-phenylenediamine 相似文献
19.
Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus 总被引:1,自引:0,他引:1
Wytske de Vries H. G. D. Niekus Hugerien van Berchum A. H. Stouthamer 《Archives of microbiology》1982,131(2):132-139
Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H+/O values and H+/NO
2
-
values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H+/2e value of 2.0. The H+/O and H+/NO
2
-
values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP
carbonylcyanide-m-chlorophenylhydrazone
- HQNO
2-n-heptyl-4-hydroxyquinoline-N-oxide
- MTPP+
methyltriphenylphosphonium cation
- TMPD
N,N,N,N-tetramethyl-p-phenylenediamine; H+/O (H+/NO
2
-
), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO
2
-
); H+/2e (q+/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor 相似文献
20.
Koyu Hon-nami Michael P. Coughlan Hiromi Hon-nami Lars G. Ljungdahl 《Archives of microbiology》1986,145(1):13-19
Clostridium thermocellum, strain JW20 (ATCC 31449) when growing in cellulose produces a cellulolytic enzyme system, that at the early stage of the fermentation is largely bound to the substrate. As cellulose is consumed the bound enzyme is released as free enzyme to the culture fluid. The bound enzyme fraction extracted with distilled water from the cellulose contains two major components, a large complex (Mr100×106) and a small complex Mr4.5×106) which were separated by gel filtration and sucrose solved by affinity chromatography into a complex that binds to the column and into a non-bindable mixture of proteins. All four fractions have endo--glucanase activity but only the two bound complexes and the free bindable complex hydrolyze crystalline cellulose with cellobiose as the main product. These three complexes are qualitatively similar in that they each contain about 20 different polypeptides (Mr values from 45,000 to 200,000) of which about ten are major components. However, the relative amounts of some of the peptides in the complexes differ. At least four polypeptides of the complexes have endo--glucanase activity.Abbreviations CM
cellulose, carboxymethyl cellulose
- CMCase
carboxymethyl cellulase cosidered endo--1,4-glucanase
- SDS
sodium dodecyl sulfate
- YAS
yellow affinity substance
- YAS-cellulose
yellow affinity substance-cellulose complex 相似文献