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1.
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
2.
Culture experiments were carried out with Acropora sp. (a branching scleractinian coral) in seawater at two pCO2 conditions (438 and 725 µatm) and two temperatures (25 and 28 °C) in order to establish the pH and temperature dependence of the boron isotopic composition of the skeleton. A clear pCO2 effect, but no temperature effect, on the coral boron isotope composition is seen. For corals cultured at normal pCO2 (438 µatm), the 11B of the skeleton was 24.0±0.2 at 25 °C, and 23.9±0.3 at 28 °C. The values of 11B measured for corals cultured at higher pCO2 (725 µatm) were lower: 22.5±0.1, and 22.8±0.1 at 25 and 28 °C, respectively. The 11B of corals cultivated at both high and normal pCO2 conditions are consistent with a dominant pH control, and are very close to that calculated from theoretical considerations. Thus, the corals do not seem to significantly alter ambient seawater for calcification with respect to pH. Co-variation between boron and carbon isotope values is explored.Communicated by: Guest Editor A. Grottoli 相似文献
3.
Leaf mesophyll protoplasts of oat (Avena sativa L.) were allowed to fix 14C-labeled bicarbonate in the absence or presence of pyridoxal phosphate (PLP), a specific inhibitor of the phosphate translocator of the inner envelope membrane of chloroplasts. The incubation was terminated by a method of rapid integrated protoplast homogenization and fractionation, and compartmented levels of label contained in sugars, phosphate esters, amino acids and organic acids were determined. The results show that the addition of PLP to a suspension of intact protoplasts causes an accumulation of phosphate esters in the chloroplasts stroma for up to 2.5 min of incubation, with a corresponding decrease in the cytosol. Prolonged treatment of protoplasts with PLP in the light resulted in a decrease of starch-associated label, combined with higher levels of labeled sugars in the cytosol, indicating a switch from phosphorolytic to hydrolytic starch degradation. Together with the determination of pool sizes of triose phosphates and of inorganic phosphate, the results demonstrate that the method employed is an important tool in investigating processes of intracellular regulation. They are discussed with respect to the permeability and possible side reactions of PLP, as well as in the light of reports on PLP action on isolated chloroplasts.Abbreviations Pi
orthophosphate
- PLP
pyridoxal 5-phosphate
- TP
triosephosphate 相似文献
4.
The enzymes coded for by two alleles at the glucuronidase structural locus (Gus) were compared in their response to pH, buffering anion, buffer molarity, ionic strength, and temperature. The heat-labile Gush gene product responded in a qualitatively similar but quantitatively reduced manner compared to the relatively heat-stable Gus
b gene product. In all buffers tested, the enzyme was most heat stable at pH 5.0. Ranking of the various buffer anions tested, according to increasing heat stabilization, was water acetate phosphate < citrate. Varying the molarity of the buffers from 0.01 to 0.6 m at pH 5.0 revealed further differences among the buffers. Increasing ionic strength exerted a destabilizing force on the protein. The half-life of the enzyme decreased by as much as a hundredfold between 71 and 75 C. The Gush/Gush genotype also results in decreased activity levels in all tissues, reportedly because of decreased synthesis. The heat inactivation curves of Gusb/Gush heterozygotes were incompatible with any theoretical curve based on the assumption that the Gusb and Gush chromosomes in the heterozygote behave in a manner similar to that seen in the homozygotes.This research was supported by a Basil O'Connor Starter Research Grant from the National Foundation—March of Dimes (R. J. M.) and by a grant from The Jane Coffin Childs Memorial Fund for Medical Research (K. H.).Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. 相似文献
5.
Yukihiko Hirai Eugene A. Permyakov Lawrence J. Berliner 《Journal of Protein Chemistry》1992,11(1):51-57
The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR. 相似文献
6.
Long-Sen Chang 《Journal of Protein Chemistry》1996,15(5):473-480
Notexin fromNotechis scutatus scutatus snake venom was subjected to Lys modification with pyridoxal 5-phosphate (PLP), and one major modified derivative was purified on a cation-exchanger SP-8HR column. The results of amino acid analysis and sequence determination revealed that only 2 Lys residues at positions 82 and 115 out of 11 Lys residues in notexin were modified. The incorporation of PLP into the protein was accompanied by the loss of 53% lethal toxicity, but the modified notexin showed an about 1.2-fold increase in enzymatic activity. However, the secondary structure of the toxin molecule did not significantly change after modification with PLP as revealed by the CD spectra, and the antigenicity of PLP derivative remained unchanged. The modified derivative retained its affinity for Ca2+, indicating that the modified Lys residues did not participate in Ca2+ binding. These results indicate that modification of Lys residues causes a differential effect on the enzymatic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one responsible for the catalytic activity and the other associated with its lethal effect. 相似文献
7.
1-Bungarotoxin (1-Bgt) fromBungarus multicinctus (Taiwan banded krait) snake venom was subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0 and the NBS derivatives were separated by high-performance liquid chromatography. The results of amino acid analysis revealed that only one Tyr residue out of 14 was modified, and the modified residue was identified to be Tyr-68 in the A chain of 1-Bgt. Spectrophotometric titration indicated that the phenolic group of Tyr-68 has apK of 10.1. Modification of Tyr-68 in the A chain caused a selective loss in lethal toxicity, but had no effect on either enzymatic or antigenic activities. The Ca2+-induced difference spectra and fluorescence study indicated that 1-Bgt possesses at least two different types of Ca2+-binding sites. However, modification of Tyr-68 in 1-Bgt did not cause any change of the Ca2+-induced difference spectra and fluorescence spectra in native toxin and the two types of Ca2+-binding sites were retained. Moreover, the affinity of Tyr-68-modified 1-Bgt for 8-anilinonaphthalene sulfonate was also unaffected in both the presence and absence of Ca2+. All of the results indicated that Tyr-68 is not involved in the Ca2+ and substrate bindings in the A chain of 1-Bgt. It is concluded that lethal toxicity is not necessarily associated with enzymatic, antigenic, and Ca2+-binding activities in 1-Bgt. 相似文献
8.
Summary O-Acetylserine sulfhydrylase is a pyridoxal 5-phosphate (PLP) dependent enzyme that catalyzes the final step of L-cysteine biosynthesis inSalmonella, viz. the conversion of O-acetyl-L-serine (OAS) and sulfide to L-cysteine and acetate. A spectrophotometric assay is available using 5-thio(2-nitrobenzoate) (TNB) as an analog of sulfide and monitoring the disappearance of absorbance at 412 nm. The enzyme catalyzes a ping pong mechanism with-aminoacrylate in Schiff base with the active site PLP as a covalent intermediate. Using data obtained from the pH dependence of kinetic parameters, the acid-base chemical mechanism and the optimum protonation state of enzyme and substrate functional groups necessary for binding has been determined. The Schiff base and the-amine of the substrate OAS are unprotonated for binding. There also appears to be a requirement for one active site general base to accept a proton from the-amine and to donate a proton to form cysteine. The enzyme also catalyzes an OAS hydrolase activity, and the pH dependence of this reaction suggests that the active site lysine that participated in the Schiff base linkage is protonated to start the second half reaction, and has a pK of about 8.2. The stereochemistry of3H-borohydride reduction of the Schiff base in free enzyme has been determined by degradation of the resulting pyridoxyllysine to pyridoxamine and measuring3H-release with apo-aspartate aminotransferase. The sequence around the active site lysine is AsnProSerPheSerValLysCysArg. 相似文献
9.
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
10.
Histatin 8 (Lys1-Phe-His-Glu-Lys5-His-His-Ser-His-Arg10-Gly-Tyr12)belongs to a group of related neutral and basic histidine richpeptides present in human salivary secretions that possess fungicidal and bactericidal activities. The conformation of thispeptide has been examined by 1H and 13C 2D-NMR in DMSO-d6, water (pH 4.0) and 40% HFA solutions. MD simulations incorporating NMR data was used to generate the solution conformations. The structures were refined by MARDIGRAS employing the RANDMARDI approach. In both DMSO-d6 and water, the peptide is seen to adopt a -pleated sheet, while HFA induces an -helix structure. The role of these structures in its mechanism of action has been explained. 相似文献
11.
Peroxides can enhance field-stimulated [3H]norepinephrine ([3H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional 2-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [3H]NE and prepared for studies of neurotransmitter release using the superfusion method. 2-Adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [3H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H2O2 (300 M) had no effect on inhibition of evoked [3H]NE release caused by the 2-adrenergic agonists. However, H2O2 (300 M) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM–1 M). In contrast, yohimbine (1 M) did not prevent the enhancement of evoked [3H]NE overflow induced by H2O2 (300 M). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional 2-adrenoceptors. 相似文献
12.
In crown-gall tumor tissue obtained from leaves of Bryophyllum daigremontianum an adenosine 3:5-cyclic phosphate (3:5-cyclic-AMP) degrading activity increases up to 2.5 fold until the fifth day after inoculation with Agrobacterium tumefaciens, declining to the value of the control in the solid tumor. Theophylline up to 1 mmol l–1 given to wounded leaves of Bryophyllum daigremontianum has no effect on the number of tumors. The effect of higher concentrations given over extended periods can be explained otherwise. Therefore it seems likely that the 3:5-cyclic-AMP phosphodiesterase (EC 3.1.4.17) has no effect on transformation and growth of crown-gall tumors in Bryophyllum daigremontianum. 相似文献
13.
R. Lohrmann 《Journal of molecular evolution》1977,10(2):137-154
Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates pnA (n 3) or P1, P2-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im
imidazole
- hist
histamine
- gly
glycine
- en
ethylenediamine
- CDI
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
- EDTA
ethylenediaminetetraacetic acid
- A
adenosine
- Pn (n = 1, 2 )
linear polyphosphate containing n phosphate residues
- pnA
adenosine 5-polyphosphate containing n phosphate residues
- ADP
adenosine 5-diphosphate
- ATP
adenosine 5-triphosphate
- AppA
P1, P2-diadenosine 5-diphosphate
- gly-pA
adenylyl-(5N)-glycine
- ImpA
adenosine 5-phosphorimidazolide
- NH2-pA
adenosine 5-phosphoramidate
- en-pA
adenylyl-(5N)-ethylenediamine
- hist (NH) - pA
adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide]
- hist(Im)-pA
adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide]
- enP1,2
phosphoramidates of ethylenediamine derived from H3PO4 and H4P2O7 相似文献
14.
Hisanori Suzuki Yasuharu Tanaka Daniela Tornese Buonamassa Benedetta Farina Enzo Leone 《Molecular and cellular biochemistry》1987,74(1):17-20
The effects of analogs of diadenosine 5,5-p1,p4-tetraphosphate (Ap4A) were examined on the ADP-ribosylation reaction of histone Hl catalysed by purified bovine thymus poly(ADP-ribose)transferase. Among the compounds tested, Ap4A and ApCH2PPPA were shown to be the most efficient inhibitors of the enzyme. From kinetic studies of their action, it appears that Ap4A and ApCH2pppA might be mixed type inhibitors.Abbreviations ADP-ribose
adenosine diphosphate ribose
- ADPRT
poly-(ADP-ribose)transferase
- Ap4A
diadenosine 5,5-p1,p4-tertraphosphate
-
Ap4A
diadenosine 5,5-p1,p4(-1,N6-ethenyl-)tetra-phosphate
-
ApAA
diadenosine 5,5-p1,p4(-N6(-1,N6-)bisethenyl-)tetraphosphate
- ApCH2pppA
diadenosine 5,5-p1,p4(-p1,p2-methylene-)tetraphosphate
- AppCH2ppA
diadenosine 5,5-p1,p4(-p2,p3methylene-)tetraphosphate
- AppNHppA
diadenosine 5,5-p1,p4(-p2,p3-amino-)tetraphosphate
- AppCHBrppA
diadenosine 5,5-p1,p4(-p2,p3-bromine methyno-)tetraphosphate
- CpCH2ppCH2PC
dicytidine 5,5-p1,p4(-p1,p2-p3,p4-bismethylene-)tetraphosphate
- ApCH2ppCH2pA
diadenosine 5,5-p1,p4(-p1,p2-p3,p4-bismethylene-)tetraphosphate. 相似文献
15.
The murine gastric mucosa possesses very high secretory type phospholipase A2 activity. Northern and Western blots indicated that the pancreatictype, sPLA2IB represents the predominant form of sPLA2 enzymes present in the gastric mucosa. Both sPLA2IB mRNA and protein in the gastric mucosa exceeded levels found in the pancreas, and in contrast to the pancreatic enzyme it was present primarily in the active state. The sPLA2IB gene is not expressed in the murine small intestine and colon. Infection by the gastritis-inducing bacteria, Helicobacter felis (H. felis) dramatically and time dependently decreased the PLA2 activity in the glandular stomach of the mouse strain, C57BL/6, sensitive to the organism, which appeared to be related to a decrease in the percentage of sPLA2IB present in the active form. This bacterial-induced reduction in PLA2 activity was not observed in BALB/c mice that fail to develop gastritis in response to H. felis infection. C57BL/6 mice do not, while BALB/c mice express, the PLA2II enzyme. The H. felisinduced reduction in sPLA2IB activity may weaken the gastric barrier by reducing the local concentration of arachidonic and linoleic acid, liberated from membrane phospholipids, the major precursors of cytoprotective prostaglandins. Data presented here suggest that both sPLA2IB and sPLA2II enzymes may contribute to the gastric response to Helicobacter infection. 相似文献
16.
Volume-sensitive taurine transport in fish erythrocytes 总被引:5,自引:0,他引:5
Daron A. Fincham Michael W. Wolowyk James D. Young 《The Journal of membrane biology》1987,96(1):45-56
Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na+-dependent -amino-acid transport system which also required Cl– for activity (apparentK
m
(taurine) 75 and 80 m;V
max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na+/Cl–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na+-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na+-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide. 相似文献
17.
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
18.
Elsebet Ø. Nielsen Marianne Aarslew-Jensen Nils Henrik Diemer Povl Krogsgaard-Larsen Arne Schousboe 《Neurochemical research》1989,14(4):321-326
The release ofd-[3H]aspartate (used as a tracer for endogenous glutamate and aspartate) was studied at high K+ (100 mM) and under ischemia in rats implanted with 0.3 mm diameter dialysis tubing through the hippocampus. The effect on thed-[3H]aspartate release of the two -aminobutyric acid (GABA) agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP) and (±)--(p-chlorophenyl)GABA (baclofen), which specifically activate GABAA and GABAB receptors, respectively, was studied. Initial experiments employing HPLC analysis showed a coincident increase in the amounts of glutamate, aspartate and the amount of radioactivity following introduction of K+ (100 mM) or a period of ischemia suggesting that thed-[3H]aspartate labels the transmitter pools of the two amino acids under the present experimental conditions. The presence of 10 mM baclofen or 10 mM THIP in the perfusion medium did not inhibit ischemia inducedd-[3H]aspartate release. On the contrary, 10 mM baclofen alone (but not 0.1 or 1 mM) in the perfusion medium induced release ofd-[3H]aspartate in a calcium dependent manner, whereas 10 mM THIP had no significant releasing effect.Special issue dedicated to Dr. Elling Kvamme 相似文献
19.
R. Lohrmann 《Journal of molecular evolution》1976,8(3):197-210
Summary Aqueous solutions of linear inorganic polyphosphates incubated in presence of Mg ions depolymerize to give trimetaphosphate. The presence of a nucleoside 5-phosphate has little influence upon the reaction. Drying the products obtained by incubating a linear polyphosphate with Mg ions in the presence of a nucleoside 5-phosphate yields nucleoside 5-polyphos-phates. The prebiological relevance of the reactions is discussed.Abbreviations Pn(n=1,2,3,)
linear polyphosphate containing n phosphate residues
- P3!
trimetaphosphate
- A
adenosine
- pnN
nucleoside 5-polyphosphate containing n phosphate residues, e.g. with N = A, n = 4
- p4N
adenosine 5-tetraphosphate
-
P
*
lpmA
pnA, (n = 1 + m); adenosine 5-polyphosphate containing n phosphate units with33p-label on terminal 1 phosphate groups 相似文献
20.
The-galactosidase fromThermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks the-peptide and an important-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg2+ bound per monomer and Mg2+ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg2+ in the-galactosidase fromEscherichia coli) probably accounts for the low level of Mg2+ binding and the consequent lack of response to Mg2+. Both Na+ and K+ also had no effect on the activity. The enzyme activity witho-nitrophenyl--D-galactopyanoside (ONPG) was very similar to that withp-nitrophenyl--D--D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to theE. coli -galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by theT. thermosulfurigenes -galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of theE. coli -galactosidase. Trp-999 is probably of the most importance. Trp-999 of theE. coli -galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of theT. thermosulfurigenes -galactosidase is different was strengthened by competitive inhibition studies. Compared toE. coli -galactosidase, D-galactonolactone was a very good inhibitor of theT. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate thatT. thermosulfurigenes -galactosidase binds the transition state differently than doesE. coli -galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature ofT. thermosulfurigenes. 相似文献