共查询到20条相似文献,搜索用时 31 毫秒
1.
N. Schilling 《Planta》1982,154(1):87-93
The de novo synthesis of maltose in spinach (Spinacia oleracea L.) was shown to be catalyzed by a maltose synthase, which converts two molecules of -d-glucose-1-phosphate (-G1P) (Km 1.5 mmol l-1) to maltose and 2 orthophosphate (Pi). This enzyme was purified 203-fold by fractionated ammonium sulfate precipitation and by column chromatography on Sepharose 6B. The addition of -G1P (15 mmol l-1) to the isolation buffer is required to stabilize the enzyme activity during the extraction and purification procedure. Molecular weight determination by gel filtration yielded a value of 95,000. -Gluconolactone, ATP and Pi are competitive inhibitors toward the substrate -G1P. The maltose synthase catalyzes an exchange of the phosphate group of -G1P with [32P] orthophosphate; this transfer reaction suggests that the synthesis of maltose occurs via a glucose-enzyme in a double displacement reaction. The physiological role of this enzyme as a starch initiator system is discussed.Abbreviations Fru
fructose
- Glc
glucose
- -G1P
-d-glucose-1-phosphate
- -G1P
-d-glucose-1-phosphate
- G6P
d-glucose-6-phosphate
This enzyme is tentatively called maltose synthase in this publication 相似文献
2.
Erika Staudacher Thomas Dalik Petra Wawra Friedrich Altmann Leopold März 《Glycoconjugate journal》1995,12(6):780-786
An 1,3-fucosyltransferase was purified 3000-fold from mung bean seedlings by chromatography on DE 52 cellulose and Affigel Blue, by chromatofocusing, gelfiltration and affinity chromatography resulting in an apparently homogenous protein of about 65 kDa on SDS-PAGE. The enzyme transferred fucose from GDP-fucose to the Asn-linkedN-acetylglucosaminyl residue of an N-glycan, forming an 1,3-linkage. The enzyme acted upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2Man3 GlcNAc2. Fucose in 1,6-linkage to the asparagine-linked GlcNAc had no effect on the activity. No transfer to N-glycans was observed when the terminal GlcNAc residues were either absent or substituted with galactose.N-acetyllactosamine, lacto-N-biose andN-acetylchito-oligosaccharides did not function as acceptors for the 1,3-fucosyltransferase.The transferase exhibited maximal activity at pH 7.0 and a strict requirement for Mn2+ or Zn2+ ions. The enzyme's activity was moderately increased in the presence of Triton X-100. It was not affected byN-ethylmaleimide.Abbreviations 1,3-Fuc-T
GDP-fucose:-N-acetylglucosamine(Fuc to Asn-linked GlcNAc)1,3-fucosyltransferase
- 1,6-Fuc-T
GDP-fucose:-N-acetylglucosamine(Fuc to Asn-linked GlcNAc) 1,6-fucosyltransferase
- PA
pyridylamino
- GnGn
GlcNAc1-2Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4GlcNAc
- GnGnF3
GlcNAc1-2Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-3)GlcNAc
- GnGnF6
GlcNAc1-2-Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc
- GnGnF3F6
GlcNAc1-2Man1-6(GlcNAc1-2Man1-3)Man1-4GlcNAc1-4(Fuc1-3)[Fuc1-6]GlcNAc
- MM
Man1-6(Man1-3)Man1-4GlcNAc1-4GlcNAc
- MMF3
Man1-6(Man1-3)Man1-4GlcNAc1-4(Fuc1-3)GlcNAc
- MMF3F6
Man1-6(Man1-3)Man1-4GlcNAc1-4(Fuc1-3)[Fuc1-6]GlcNAc 相似文献
3.
Nobuyoshi Esaki Mutsumi Watanabe Tatsuo Kurihara Kenji Soda 《Archives of microbiology》1994,161(2):110-115
-Dialkylamino acid aminotransferase was found in various fungi; this is the first evidence for the occurrence of the enzyme in eukaryotes. The enzyme was purified from Fusarium solani and shown to be composed of four subunits with an identical molecular weight of 42,000. -Aminoisobutyrate and cycloleucine served as amino donors, and pyruvate, -ketobutyrate, -ketovalerate, -ketoisovalerate, and glyoxylate as amino acceptors. The K
m values for -aminoisobutyrate and -ketobutyrate were 28 and 0.3 mM, respectively. -Ketobutyrate inhibited the enzyme noncompetitively with -aminoisobutyrate, and showed K
i value of 8 mM. The significant inhibitory effect of l-cycloserine was observed, but d-cycloserine did not inhibit the enzyme. The pH and temperature optima for transamination of -aminoisobutyrate with pyruvate were about 8.0 and 60°C, respectively. Despite the production of this enzyme by the mesophile, the enzyme was thermostable; it retained its full activity upon heating at 60°C for 30 min.Abbreviations ACPC
1-aminocyclopropane-1-carboxylic acid
- AIB
-aminoisobutyrate
- PLP
pyridoxal 5-phosphate 相似文献
4.
The activities of -2-l-fucosyltransferase and -3-l-fucosyltransferase were measured in human platelets and leucocytes from normal donors, -2-l-Fucosyltransferase was found in platelets but not in leucocytes. In contrast -3-l-fucosyltransferase was not detected in platelets but was present in leucocytes where it was demonstrated in the neutrophil, monocyte and lymphocyte fractions. 相似文献
5.
Summary A simple E.COSY type technique is described for measurement of two-bond JCOH coupling constants in proteins that are uniformly enriched with13C. The method has been used to measure2JCOH for 132 residues in the proteins calmodulin and staphylococcal nuclease having non-overlapping H–C correlations. Measured2JCOH coupling constants fall in the 0 to –9.5 Hz range. A separate experiment, measuring the accuracy of these values, indicates a root-mean-square error of 1 Hz. Comparison of the J couplings with the dihedral back bone angles from crystallographic studies confirms a weak but statistically significant correlation between the dihedral angle and the magnitude of2JCOH, but also indicates that parameters other than have a significant effect on the value of the coupling. 相似文献
6.
Marco Crisma Vania Monaco Fernando Formaggio Claudio Toniolo Clifford George Judith L. Flippen-Anderson 《Letters in Peptide Science》1997,4(4-6):213-218
An X-ray diffraction analysis of the [Fmoc0,TOAC4,8, Leu-OMe11] analogue of thelipopeptaibol antibiotic trichogin A IV shows that the undecapeptide isfolded in a right-handed, mixed /310-helix. The helicalmolecules are connected in a head-to-tail arrangement along the b-axisthrough C=O···H-N intermolecular H-bonding. Thispacking mode generates a hydrophobic cavity where the FmocN-protecting groups are accommodated. The distances andangles between the nitroxide groups of the two TOAC residues, separated byone turn of the -helix, have been determined. 相似文献
7.
H. A. J. Struijker-Boudier M. W. J. Messing H. van Essen 《Molecular and cellular biochemistry》1996,157(1-2):239-244
The goal of this study was to determine the functional distribution of 1- and 2-adrenoceptors in the striated muscle microcirculation. Experiments were performed in intact conscious spontaneously hypertensive rats (SHR) that were provided with a dorsal microcirculatory chamber to allow microvascular diameter measurements. Administration of selective 1- and 2-agonists, phenylephrine and azepexole, respectively, induced different patterns of microvascular constriction. 1-Adrenoceptor stimulation showed a preferential constriction of large arteries and venules. The entire arteriolar microvasculature was sensitive to 2-adrenoceptor stimulation, whereas the venular vessels did not respond to azepexole. The selective 1- and 2-antagonists prazosin and yohimbine showed patterns of vasodilator activity comparable to those of the corresponding agonists. The specificity of the drug-induced effects was verified by comparing their effects with those of graded hemorrhage, a non-pharmacological method for blood pressure lowering. In the range of blood pressure decreases comparable to that obtained by -adrenoceptor antagonists, graded hemorrhage did not influence microvascular diameters. These results show a differential functional distribution of 1- and 2-adrenoceptors along the microvascular tree in striated muscle of conscious SHR. 相似文献
8.
Rajendra Prasad Roy Parimala Nacharaju Ronald L. Nagel A. Seetharama Acharya 《Journal of Protein Chemistry》1995,14(2):81-88
Interspecies hybrids of HbA and Hb from mouse C57BL/10 [
2
M
2
H
and
2
H
2
M
(H=human, M=mouse)], representing 19 and 27 sequence differences per dimers (as compared with human dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O2 binding. The intrinsic O2 affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid
2
M
2
H
has high O2 affinity (compared to either human or mouse Hb), while the interspecies hybrid
2
H
2
M
exhibits a very low O2 affinity. These results suggest that the mouse chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse and chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse chain. DPG binds the tetramer in the central cavity formed by the two subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the chain present in the tetramer. The mouse chain reduces considerably the DPG right shift of the O2 affinity of the human-chain containing hybrid. Sequence analysis suggest that perturbations of the
1
1 (not the
1
2) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse chains in transgenic mouse expressing human
S chains. 相似文献
9.
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
10.
Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The 2A-adrenoceptor (2AAR), which yielded an efficacious and weak [35S]GTPS binding response by respectively G o and G i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G o and G i3 proteins, and reciprocally, were permuted. Activation of the chimeric G o / i3 protein was highly efficient whereas the G i3 / o protein yielded a weak stimulation. These [35S]GTPS binding responses were not different from their parental wild-type G o and G i3 proteins. Similar results were obtained with an 2AAR carrying a facilitating Thr373Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G o protein amino acids do not constitute a major 2AAR interaction domain for G protein activation. 相似文献
11.
M. Ando E. Omura 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(1):64-69
Dose-response relationships of catecholamines on seawater eel intestinal ion transport were obtained; the potency order being (-)adrenalin>(-)noradrenalin=clonidine>(±)noradrenalin (racemic form of noradrenalin)>(-)phenylephrine>dopamine>(±)isoproterenol, indicating that 2 are more potent than 1- or -agonists. In addition, the effects of adrenalin were completely blocked by yohimbine (2-antagonists) but not by prazosin (1-antagonists) or propranolol (-antagonist). These results indicate the existence of an 2-receptor in the seawater eel intestine. Adrenalin may activate the 2-receptor physiologically, since adrenalin is the most potent stimulant and is the predominant catecholamine in American eel plasma (Hathaway and Epple 1989). Presumably ion and water absorption across the seawater eel intestine will be maintained by adrenalin. From the structure and the action of various agents used in the present study, structure-activity relationships of catecholamines are considered: hydroxyl groups on the benzene ring (catechol) seem to be essential for the 2-action in the seawater eel intestine and the presence of OH and CH3 on -carbon and amide, respectively, seems to potentiate the 2-action.Abbreviations ACh
acetylcholine
- AD
adrenalin
- CONH
2-cyclooctyl-2-hydroxyethylamine; n-methyltransferase
- DA
dopamine
- 5-HT
serotonin
- IBMX
3-isobutyl-1-methylxanthine
-
I
sc
short-circuit current
- MCh
methacholine
- NA
noradrenalin
- PD
transepithelial potential difference
- PNMT
phenylethanolamine N-methyltransferase
-
R
t
tissue resistance 相似文献
12.
2-Macroglobulin (2M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the 2M sequence. In the 3D-structure of 2M, TGF- occupies the 2M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary 2M–protease complexes (2 mol protease/mol 2M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to 2M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary 2M–trypsin complex (1 mol trypsin/mol 2M) binds increased amounts of TGF-1, compared with native 2M, while ternary 2M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary 2M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native 2M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the 2M central cavity; however, 2M–plasmin complex also bound increased amounts of A, compared with native 2M. We conclude that A accesses its binding site, in 2M, from outside the 2M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of 2M, but also in the 3D-structure. 相似文献
13.
Morishita F Shimada A Fujimoto M Katayama H Yamada K 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(7):533-540
The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by -adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·l-1 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3,5-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol·l-1 and naphazoline at 1 mol·l-1 inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate in the cells in both the presence and the absence of isoproterenol, a -adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3,5-monophosphate, but only when they were present at relatively high concentrations (above 100 mol·l-1). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3,5-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 g·ml-1) for 15 h or treatment with 100 nmol·l-1 yohimbine (an 2-adrenergic antagonist) inhibited the effects of the -adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate, but prazosin (an 1adrenergic antagonist) at 100 nmol·l-1 was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase, which occurs as result of stimulation of a pathway that involves 1adrenergic and a inhibitory GTP-binding protein.Abbreviations A-kinase
cAMP-dependent protein kinase
- BSS
balanced salts solution
- CaM
calmodulin
- cAMP
cyclic adenosine-3,5-monophosphate
- Clo
clonidine
- EDTA
ethylenediaminetetra-acetic acid
- G-protein
GTP-binding protein
- HEPES
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
- IBMX
3-isobutyl-1-methylxanthine
- IP3
inositol 1,4,5-trisphosphate Mex, methoxamine
- MSH
melanocyte-stimulating hormone
- Nap
naphazoline
- NE
norepinephrine
- Oxy
oxymetazoline
- Phe
phenylephrine
- PTX
pertussis toxin 相似文献
14.
This review summarizes our experiments on the significance of the -subunit in the functional expression of Na+/K+-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na+/K+-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH2-terminal domain to the extracellular COOH-terminus: the NH2-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na+/K+-ATPase by Na+/K+- and H+/K+-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na+/K+-ATPase -subunit and from Ile89 to the COOH–terminus of the H+/K+-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na+/K+-ATPase. 相似文献
15.
The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl2 two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[3H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE
N-carbobenzoxy-L-tyrosine p-nitrophenylester
- Cyt oxidase
Cytochrome oxidase
- ER
endoplasmic reticulum
- EDTA
ethylenediaminetetraacetic acid
- GA3
gibberellic acid
- IDPase
inosine diphosphatase
- K+-ATPase
pH 6.5 K+-stimulated adenosine triphosphatase
- MES
2-(N-morpholino)ethanesulfonic acid
- MOPS
3-(N-morpholino)propanesulfonic acid
- NADH: Cyt c reductase
cyanide-insensitive NADH-linked cytochrome-c reductase
- RER
rough endoplasmic reticulum
- Tris
tris-(hydroxymethyl)-aminomethane 相似文献
16.
W. -M. Weber B. Dannenmaier W. Clauss 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(2):153-159
The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm-2) with an initial short-circuit current of 29.0±2.9 A·cm-2. Removal of Na+ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l-1) added to the basolateral solution, depressed the short-circuit current completely. The Na+ current saturated at a concentration of 90 mmol Na+·l-1 in the apical solution (K
M=11.2±1.8 mmol·l-1). Amiloride (100 mol·l-1) on the apical side inhibited ca. 40% of the Na+ current and indicated the presence of Na+ channels. The dependence of Na+ current on the amiloride concentration followed Michaclis-Menten kinetics (K
i=2.9±0.4 mol·l-1). The amiloride analogue benzamil had a higher affinity to the Na+ channel (K
i=0.7±0.2 mol·l-1). Thus, Na+ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na+·l-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na+ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l-1) and isobutylmethylxanthine (1 mmol·l-1) nearly doubled short-circuit current and increased amiloride-sensitive Na+ currents by 50%. By current fluctuation analysis we estimated single Na+ channel current (2.7±0.9 pA) and Na+ channel density (3.6±0.6 channels·m-2) under control conditions. After cyclic adenosine monophosphate stimulation Na+ channel density increased to 5.4±1.1 channels·m-2, whereas single Na+ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na+ channel, and show the similarities and differences to vertebrate Na+ channels. Whereas the channel properties are different from the classical vertebrate Na+ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na+ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na+ channels.Abbreviations
slope of the background noise component
- ADH
antidiuretic hormone
- cAMP
cyclic adenosine monophosphate
-
f
frequency
-
f
c
coner frequency of the Lorentzian noise component
- Hepes
N-hydroxyethylpiperazine-N-ethanesulphonic acid
- BMX
isobutyl-methylxanthine
-
i
Na
single Na+ channel current
-
I
Na
max, maximal inhibitable Na+ current
-
I
SC
short circuit current
-
K
i
half maximal blocker concentration
-
K
M
Michaelis constandard error of the mean
-
S
(f)
power density of the Lorentzian noise component
-
S
0
plateau value of the Lorentzian noise component
- TMA
tetramethylammonium
- Trizma
TRIS-hydroxymethyl-amino-methane
-
V
max
maximal reaction velocity
-
V
T
transepithelial potential
-
K
half maximal blocker concentration 相似文献
17.
Subfamilies of voltage-activated K+ channels (Kv1-4) contribute to controlling neuron excitability and the underlying functional parameters. Genes encoding the multiple subunits from each of these protein groups have been cloned, expressed and the resultant distinct K+ currents characterized. The predicted amino acid sequences showed that each subunit contains six putative membrane-spanning -helical segments (S1-6), with one (S4) being deemed responsible for the channels' voltage sensing. Additionally, there is an H5 region, of incompletely defined structure, that traverses the membrane and forms the ion pore; residues therein responsible for K+ selectivity have been identified. Susceptibility of certain K+ currents produced by the Shaker-related subfamily (Kv1) to inhibition by -dendrotoxin has allowed purification of authentic K+ channels from mammalian brain. These are large (Mr 400 kD), octomeric sialoglycoproteins composed of and subunits in a stoichiometry of ()4()4, with subtypes being created by combinations of subunit isoforms. Subsequent cloning of the genes for 1, 2 and 3 subunits revealed novel sequences for these hydrophilic proteins that are postulated to be associated with the subunits on the inner side of the membrane. Coexpression of 1 and Kv1.4 subunits demonstrated that this auxiliary protein accelerates the inactivation of the K+ current, a striking effect mediated by an N-terminal moiety. Models are presented that indicate the functional domains pinpointed in the channel proteins. 相似文献
18.
We examined the effects of changing KCl concentration on the secondary structures of -actinins using circular dichroism (CD), 1,1-bis(4-anilino) naphthalene-5,5-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mm KH2PO4, pH 7.5, increasing KCl from 0 to 120 mm led to decreases in -helix conformation for brain, platelet and heart -actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mm KCl, 0.65 mm calcium produced small changes in the CD spectra of both brain and platelet -actinin, but had no effect on heart -actinin. bisANS fluorescence of all three -actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mm KCl increasing concentrations of Ca2+ or Mg2+ did not have significant effects on the bisANS fluorescence of any -actinin. Digestion of brain, platelet and heart -actinins with -chymotrypsin showed an increase of proteolytic susceptibility in 120 mm KCl. These experiments also showed that increasing the concentration of Ca2+ or Mg2+ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mm KCl. The results suggest that -actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different -actinins. 相似文献
19.
Hirotaka Yamamoto Yoichi Kaneko David Vandermulen Donna Kersey Edward Mkrdichian Leonard Cerullo Jan Leestma Joseph R. Moskal 《Glycoconjugate journal》1995,12(6):848-856
The expression of CMP-NeuAc: Gal1,4GlcNAc 2,6 sialyltransferase (2,6-ST) [EC 2.4.99.1] and glycoproteins bearing 2,6-linked sialic acids were examined in primary human brain tumours and cell lines. 79% (19/24) of the meningiomas expressed 2,6-ST mRNA, 42% (10/24) of which showed very high expression. 2,6-ST mRNA expression was undetectable in normal brain tissue. In contrast, only 1/13 of the gliomas examined expressed detectable 2,6-ST mRNA. Metastases to the brain did not express measurable amounts of 2,6-ST mRNA. Less expression was found in malignant (i.e., anaplastic) compared to benign (i.e. meningothelial) meningiomas. Two-dimensional SDS-PAGE of glioma and meningioma proteins, followed bySambucus nigra lectin staining, revealed the presence of a glycoprotein bearing 2,6-linked sialic acids,M
r=53 kDa and a pI=7.0 (MEN-1) that appeared in all seven of the meningiomas examined, but was expressed at barely detectable levels, if at all, in seven out of the seven glioblastomas examined. Thus, decreased 2,6-ST expression may play a role in the aggressive nature of anaplastic meningiomas, but appears to be virtually absent in all tumours of glial origin. 相似文献
20.
The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n
H
-values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH3–CH2-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA
Coenzyme A
- Tris
Tris(hydroxymethyl)aminomethane hydrochloride
- DTNB
5,5-dithiobis-(2-nitrobenzoic acid)
- IPM
-Isopropylmalate
- KIV
-Ketoisovalerate
Prepared from doctoral thesis of the University of Göttingen 1973 相似文献