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1.
Summary Amounts and temporal changes of the release of the tracer ions K+ (86Rb+),22Na+, and36Cl– as well as of H+ in the course of action potentials inAcetabularia have been recorded. New results and model calculations confirm in quantitative terms the involvement of three major ion transport systemsX in the plasmalemma: Cl– pumps, K+ channels, and Cl– channels (which are marked in the following by the prefixes,P, K andC) with their equilibrium voltages
X
V
e
and voltage/time-dependent conductances, which can be described by the following, first approximation. Let the maximum (ohmic) conductance of each of the three populations of transporter species be about the same (P
L,
KL,C
L=1) but voltage gating be different: the pump (
p
V
e
about –200 mV) being inactivated (open,oclosed,c) at positive going transmembrane voltages,V
m; the K+ channels (K
V
e about –100 mV) are inactivated at negative goingV
m; and the Cl– channels (C
V
e: around 0 mV), which are normally closed (c) at a restingV
m (nearPVe) go through an intermediate open (o) state at more positiveV
m before they enter a third shut state (s) in series. Model calculations, in which voltage sensitivities are expressed by the factorf=exp(V
mF/(2RT)), simulate, the action potential fairly well with the following parameters (PKco10/f ks–1,PKoc1000·f ks–1,KKco200·f ks–1,Kkoc2/f ks–1,cKco500·f ks–1,CKoc5/f ks–1,CKso0.1/f ks–1,Ckos20·f ks–1). It is also shown that the charge balance for the huge transient Cl– efflux, which frequently occurs during an action potential, can be accounted for by the observation of a corresponding release of Na+. 相似文献
2.
Putrescine carbamoyltransferase (PutCT) has been postulated to function in the synthesis of putrescine (Put) from an N-carbamoylputrescine (NCPut) intermediate in plants. In pea, PutCT activity was associated entirely with ornithine carbamoyltransferase (OCT) protein, which was purified to homogeneity using an immobilized transition-state analog inhibitor (
N-(phosphonacetyl)-L-ornithine). No evidence for a separate PutCT enzyme, similar to that in Streptococcus [15], or PutCT activity associated with a putrescine synthase-type multifunctional enzyme [13] was found. OCT carried out the carbamoylation of Put and other diamine and polyamine substrates inefficiently and at non-physiological pH (Put carbamoylation: pH 10.8 optimum, Vmax 0.11 kat/mg protein, Km=6.7 mM for Put and 1.0 mM for carbamoyl-P), when compared with ornithine carbamoylation (pH 8.5 optimum, Vmax=313.9 kat/mg protein, Km=4.4 mM for ornithine and 0.6 mM for carbamoyl-P). Different subcellular compartmentation of PutCT activity (chloroplast) and the NCPut substrate (cytosol), coupled with a thermodynamically-unfavorable reverse reaction (i.e., Put synthesis from NCPut), suggest that the OCT-associated PutCT activity does not significantly contribute to in vivo Put synthesis in plants.Abbreviations ADC
Arginine Decarboxylase
- Agm
Agmatine
- AIH
Agmatine Iminohydrolase
- Cad
Cadaverine
- Cit
Citrulline
- CP
Carbamoyl-P
- Dap
1,3-Diaminopropane
- Dns-
Dansyl-
- NCPut
N-Carbamoylputrescine
- OCT
Ornithine Carbamoyltransferase
- Put
Putrescine
- PutCT
Putrescine Carbamoyltransferase
- Spd
Spermidine
- Spm
Spermine 相似文献
3.
Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers 总被引:10,自引:0,他引:10
The substrate dependence and product inhibition of three different fructokinases and three different hexokinases from growing potato (Solanum tuberosum L.) tubers was investigated. The tubers contained three specific fructokinases (FK1, FK2, FK3) which had a high affinity for fructose K
m=64, 90 and 100 (M) and effectively no activity with glucose or other hexose sugars. The affinity for ATP (K
m=26, 25 and 240 M) was at least tenfold higher than for other nucleoside triphosphates. All three fructokinases showed product inhibition by high fructose (K
i=5.7, 6.0 and 21 mM) and were also inhibited by ADP competitively to ATP. Sensitivity to ADP was increased in the presence of high fructose, or fructose-6-phosphate. In certain conditions, the K
i (ADP) was about threefold below the K
m (ATP). All three fructokinase were also inhibited by fructose-6-phosphate acting non-competitively to fructose (K
i=1.3 mM for FK2). FK1 and FK2 showed very similar kinetic properties whereas FK3, which is only present at low activities in the tuber but high activities in the leaf, had a generally lower affinity for ATP, and lower sensitivity to inhibition by ADP and fructose. The tuber also contained three hexokinases (HK1, HK2, HK3) which had a high affinity for glucose (K
m=41, 130 and 35 M) and mannose but a poor affinity for fructose (K
m=11, 22 and 9 mM). All three hexokinases had a tenfold higher affinity for ATP (K
m=90, 280 and 560 M) than for other nucleoside triphosphates. HK1 and HK2 were both inhibited by ADP (K
i=40 and 108 M) acting competitively to ATP. HK1, but not HK2, was inhibited by glucose-6-phosphate, which acted non-competitively to glucose (K
i=4.1 mM). HK1 and HK2 differed, in that HK1 had a narrower pH optimum, a higher affinity for its substrate, and showed inhibition by glucose-6-phosphate. The relevance of these properties for the regulation of hexose metabolism in vivo is discussed.Abbreviations FK
fructokinase
- Fru6P
fructose-6-phosphate
- Glc6P
glucose-6-phosphate
- HK
hexokinase
- NTP
nucleoside triphosphate
- Pi
inorganic phosphate
- UDPGlc
uridine-5-diphosphoglucose
This work was supported by the Deutsche Froschungsgemeinschaft (SFB 137). We are grateful to Professor E. Beck (Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, FRG) for providing laboratory facilities. 相似文献
4.
Wilson IB 《Glycoconjugate journal》2001,18(6):439-447
Recently, it has been found that plants, including tomato (Lycopersicon esculentum), express the Lewis-a epitope, Gal1,3(Fuc1,4)GlcNAc, on some N-glycans. By searching the EST database, it was possible to identify a tomato cDNA encoding a protein, designated FucTC, of 413 amino acids with homology to plant and mammalian 1,3/4-fucosyltransferases. The cDNA was expressed in Pichia pastoris and the recombinant enzyme was found to transfer fucose from GDP-Fuc (Km 16 M) to lacto-N-tetraose (Gal1,3GlcNAc1,3Gal1,4Glc; Km 80 M) as well as to 1,3- and 1,4-galactosylated N-glycans. It is concluded that FucTC is responsible for the biosynthesis of Lewis-a on N-glycans in tomato. 相似文献
5.
Oxygen protection of nitrogenase in Frankia sp. HFPArI3 总被引:11,自引:0,他引:11
O2 protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O2 uptake and acetylene reduction rates in response to substrate concentration. O2 of NH
4
+
-grown cells showed an apparent K
m O2 of approximately 1M O2. In N2-fixing cultures a second K
m O2 of about 215 M O2 was observed. Thus, respiration remained unsaturated by O2 at air-saturation levels. In vivo, the apparent K
m for acetylene was more than 10-fold greater than reported in vitro values. These data were inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3. 相似文献
6.
The effects of temperature on the dark relaxation kinetics of nonradiative energy dissipation in photosystem II were compared in lettuce (Lactuca sativa L.) chloroplasts and leaves of Aegialitis annulata R. Br. After high levels of violaxanthin de-epoxidation in the light, Aegialitis leaves showed a marked delay in the dark relaxation of nonradiative dissipation, measured as non-photochemical quenching (NPQ) of photosystem II chlorophyll a fluorescence. Aegialitis leaves also maintained a moderately high adenylate energy charge at low temperatures during and after high-light exposure, presumably because of their limited carbon-fixation capacity. Similarly, dark-sustained NPQ could be induced in lettuce chloroplasts after de-epoxidizing violaxanthin and light-activating the ATP synthase. The duration and extent of dark-sustained NPQ were strongly enhanced by low temperatures in both chloroplasts and leaves. Further, the NPQ sustained at low temperatures was rapidly reversed upon warming. In lettuce chloroplasts, low temperatures sharply decreased the ATP-hydrolysis rate while increasing the duration and extent of the resultant trans-thylakoid proton gradient that elicits the NPQ. This was consistent with a higher degree of energy-coupling, presumably due to reduced proton diffusion through the thylakoid membrane at the lower temperatures. The chloroplast adenylate pool was in equilibrium with the adenylate kinase and therefore both ATP and ADP contributed to reverse coupling. The low-temperature-enhanced NPQ quenched the yields of the dark level (Fo) and the maximal (Fm) fluorescence proportionally in both chloroplasts and leaves. The extent of NPQ in the dark was inversely related to the efficiency of photosystem II, and very similar linear relationships were obtained over a wide temperature range in both chloroplasts and leaves. Likewise, the dark-sustained absorbance changes, caused by violaxanthin de-epoxidation (A508nm) and energy-dependent light scattering (A536nm) were strikingly similar in chloroplasts and leaves. Therefore, we conclude that the dark-sustained, low-temperature-stimulated NPQ in chloroplasts and leaves is apparently directly dependent on lumen acidification and chloroplastic ATP hydrolysis. In leaves, the ATP required for sustained NPQ is evidently provided by oxidative phosphorylation in the mitochondria. The functional significance of this quenching process and implications for measurements of photo-protection versus photodamage in leaves are discussed.Abbreviations and Symbols A
antheraxanthin
- Chl
chlorophyll
- DPS
de-epoxidation state of the xanthophyll cycle, ([Z+A]/[V+A+Z])
- F, F
steady-state fluorescence in the absence, presence of thylakoid energization
- Fo, Fo
dark fluorescence level in the absence, presence of thylakoid energization
- Fm, Fm
maximal fluorescence in absence, presence of thylakoid energization
- NPQ
nonphotochemical quenching (Fm/Fm)–1
- V
violaxanthin
- Z
zeaxanthin
- NRD
nonradiative dissipation
- PFD
photon flux density
-
[2ATP+ADP]
- pH
trans-thylakoid proton gradient
- S
pH-dependent light scattering
- PSII
(Fm–F)/Fm, photon yield of PSII photochemistry at the actual reduction state in the light or dark
-
[ATP+ADP+AMP]
We thank Connie Shih for skillful assistance in growing plants and for conducting HPLC analyses. Support from an NSF/USDA/DOE postdoctoral training grant to A.G. is gratefully acknowledged. A.G. also wishes to thank Prof. Govindjee for valuable discussions. C.I.W.-D.P.B. Publication No. 1197. 相似文献
7.
Zyakun A. M. Kosheleva I. A. Zakharchenko V. N. Kudryavtseva A. I. Peshenko V. A. Filonov A. E. Boronin A. M. 《Microbiology》2003,72(5):592-596
The study deals with a comparative analysis of the relative abundances of the carbon isotopes 12C and 13C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (13C = –44.6 ± 0.2) were characterized by the values of 13CCO
2 = –50.2 ± 0.4, 13Cbiom = –46.6 ± 0.4, and 13Cexo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (13C = –21 ± 0.4) were characterized by the isotope effects 13CCO
2 = –24.1 ± 0.4, 13Cbiom = –19.2 ± 0.4, and 13Cexo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed. 相似文献
8.
Hubert Felle 《Planta》1981,152(6):505-512
In the aquatic liverwort Riccia fluitans, membrane depolarization (m), change in membrane conductance (gm), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of 14C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (m, gm) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of 14C-labeled amino acids display three components: (I) A saturable high-affinity component with Ks-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given Ks-value. Uptake of 14C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations m
membrane potential (mV)
- Eco
equilibrium potential (mV) of the transport system
- gm
membrane (slope) conductance (Sm-2)
- gm
change in gm
- I-V curve
current-voltage curve
- AIB
-aminoisobutytric acid
- -AB
-aminobutyric acid 相似文献
9.
I. V. K. Subrahmanya Sastry G. Pavan Kumar M. Chidambaram 《Bioprocess and biosystems engineering》1994,10(3):127-130
The effect of time delay in specific growth rate () on the periodic operation of bioreactors with input multiplicities is theoretically analyzed for productivity improvement. A periodic rectangular pulse is applied either in feed substrate concentration (Sf) or in dilution rate (D). Periodic operation under feed substrate concentration cycling gives improvement in productivity at lower value of ¯Sf of the two steady-state multiplicities of Sf only when the time delay in is larger. Whereas the larger value of ¯Sf gives improvement in average productivity for all values of time delay. Dilution rate (D) cycling gives an improvement in average productivity particularly for larger time delay in . This improvement in average productivity is obtained only at smaller value of dilution rate out of the two steady-state input multiplicities of D.List of Symbols D 1/h
dilution rate
- F
memory function
- g
dummy variable
- Ki g/l
substrate inhibition constant
- Km g/l
substrate saturation constant
- P g/l
product concentration
- Pm g/l
product saturation constant
- Q g/(hl)
product cell produced per unit time
- S g/l
substrate concentration
- Sf g/l
feed substrate concentration
- Sf,p g/l
feed substrate concentration during fraction of a period
- X g/l
biomass concentration
- YX/S g/g
cell mass yield
- w
variable either S or Z
- Z g/l
weighted average of substrate concentration
Greek Letters
1/h
time delay parameter
-
1
,
2
product yield parameters, g/g and 1/h
-
pulse width expressed as a fraction of a period
-
1/h
specific growth rate
-
m 1/h
maximum specific growth rate
- h
period of oscillation
- –
average value 相似文献
10.
S. Dwivedi D. P. Singh 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1985,70(4):337-337
Summary The inheritance of the dwarf plant type was studied in blackgram (V. mungo (L.) Hepper). Type 9 has erect plant type with normal internode length. The mutant line, EMSD has reduced internode length. The F1, F2 and F3 generations of a cross between Type 9 and EMSD and its reciprocal were studied. The extreme dwarf plant type appeared to be governed by a single recessive gene, dw
1
dw
1 with no cytoplasmic effect.Part of Ph.D. Thesis submitted by the first author 相似文献
11.
Measurement of the light response of photosynthetic CO2 uptake is often used as an implement in ecophysiological studies. A method is described to calculate photosynthetic parameters, such as the maximum rate of whole electron transport and dissimilative respiration in the light, from the light response of CO2 uptake. Examples of the light-response curves of flag leaves and ears of wheat (Triticum aestivum cv. ARKAS) are shown.Abbreviations and symbols A
net photosynthesis rate
-
D
1
rate of dissimilative respiration occurring in the light
-
f
loss factor
-
I
incident PPFD
-
I
effective absorbed PPFD
-
J
rate of whole electron transport
-
J
m
maximum rate of whole electron transport
-
p
c
intercellular CO2 partial pressure
- PPFD
photosynthetic photon flux density
-
q
effectivity factor for the use of light (electrons/quanta)
-
absorption coefficient
-
I
*
CO2 compensation point in the absence of dissimilative respiration (bar)
-
II
conversion factor for calculation of CO2 uptake from the rate of whole electron transport
-
convexity factor
Gas-exchange rates relate to the projective area and are given in mol·m-2·s-1. Electron-transport rates are given in mol electrons·m-2·s-1; PPFD is given in mol quanta·m-2·s-1. 相似文献
12.
Peter v. d. Veen Michel J. A. Flipphi Alphons G. J. Voragen Jaap Visser 《Archives of microbiology》1991,157(1):23-28
The induction of arabinases in Aspergillus niger N400 was studied on different simple and complex carbon sources. Sugar beet pulp was found to be an inducer of three arabinan degrading enzymes (-l-arabinofuranosidase A, -l-arabinofuranosidase B and endoarabinase). These enzymes were purified from A. niger culture fluid after growth of the fungus in medium employing sugar beet pulp as the carbon source and were characterised both physico-chemically (Mw 83 000, 67 000, 43 000 Da and, pI 3.3, 3.5 and 3.0 for -l-arabinofuranosidases A and B and endo-arabinase, respectively) and kinetically (K
m on p-nitrophenyl--l-arabinofuranoside 0.68 and 0.52 mM for -l-arabinofuranosidases A and B, resp.; K
m on sugar beet arabinan 0.24 and 3.7 g/l for -l-arabinofuranosidase B and endoarabinase, resp.). The amino acid compositions of the three enzymes were determined also. The enzymic properties were compared with those of arabinases purified from a commerical A. niger enzyme preparation. Differences were found though the kinetic data suggest considerable similarity between the enzymes from the different sources. Antibodies raised in mice against the three enzymes were found to be highly specific and no crossreactivity with other proteins present in culture filtrates was observed. A mixture of these antibodies has been used to analyze specific induction of these individual enzymes on simple and complex substrates by Western blotting.Abbreviation PNA
p-nitrophenyl--l-arabinofuranoside 相似文献
13.
Microsomes prepared from equine submandibular glands and incubated with tritium-labelled AcCoA incorporated acid-insoluble radioactivity in a manner dependent on time, protein, membrane integrity and AcCoA concentration, with incorporation being optimal at 37°C and pH 6.6. Under the experimental conditions used a K
M of 32.1 M for AcCoA and a Vmax of 1.2 pmol/mg protein x min was obtained. The incorporation of acid-insoluble radioactivity was also inhibited by CoA in a competitive manner (K
i=240 M), as well as by para-chloromercuribenzoate, 3-dephospho-CoA, 5-IDP, 5-ADP, ß-NAD and 4,4-diisothiocyanatostilbene-2,2-disulfonate. We demonstrate here that this incorporation of radioactivity into endogenous sialic acid is due to the action of an AcCoA:sialate-4-O-acetyltransferase [EC 2.3.1.44]. Radio thin-layer chromatography analyses of propionic acid-released sialic acids showed that the incorporation of radioactivity correlated with the formation of a radiolabelled species that co-migrated with authentic Neu4,5Ac2. Saponification experiments using NaOH, mouse hepatitis virus strain S and Influenza C/JJ/50 virus also showed that the transfer of [3H]acetyl groups from [3H]AcCoA to endogenous sialic acid acceptors was occurring exclusively at carbon 4 of the pyranose ring. 相似文献
14.
Bent Vestergaard-Bogind Per Stampe Palle Christophersen 《The Journal of membrane biology》1987,95(2):121-130
Summary The conductance of the Ca2+-activated K+ channel (g
K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V
m
) and extracellular K+ concentration ([K+]ex). ATP-depleted cells, with fixed values of cellular K+ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K+ channels, to buffer-free salt solutions with given K+ concentrations. Outward-current conductances were calculated from initial net effluxes of K+, correspondingV
m
, monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E
K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of42K at (V
m
–E
K)0, using a single-file flux ratio exponent of 2.7. Within a [K+]ex range of 5.5 to 60mm and at (V
m
–E
K) 20 mV a basic conductance, which was independent of [K+]ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm2 between 0 and –100 mV. As (V
m
–E
K) decreased from 20 towards zero mVg
K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm2. The zero-current conductance was not significantly dependent on [K+]ex (30 to 156mm) corresponding toV
m
(–50 mV to 0). A further increase ing
K(Ca) symmetrically aroundE
K is suggested as (V
m
–E
K) becomes positive. Increasing the extracellular K+ concentration from zero and up to 3mm resulted in an increase ing
K(Ca) from 50 to 70 S/cm2. Since the driving force (V
m
–E
K) was larger than 20 mV within this range of [K+]ex this was probably a specific K+ activation ofg
K(Ca). In conclusion: The Ca2+-activated K+ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel. 相似文献
15.
Suzanne M. Loret Pierre E. Devos 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(7):651-657
Summary The hydrolysis of glucose-6-phospate in the digestive gland of the crab Carcinus maenas is carried out by an aspecific phosphatase. This enzyme possesses the following features: (1) insensitivity to acid treatment; (2) absence of inhibition when exposed to citrate at low pH; (3) similar affinity for G6P as the acid phosphatase for Na--glycerophosphate (K
m 2.3 and 2.0 mM, respectively). Glucose-6-phosphate and Na--glycerophate hydrolysis reactions seem to be catalysed by the same enzyme, since both activities exhibit the same distribution in a subcellular fractionation of the gland. Furthermore, as these activities are principally recovered in the subcellular fraction enriched in calcospherites (or calcium phosphate granules), it is proposed that the aspecific G6P-phosphohydrolase could play a major role in the formation of these granules. The phosphorylation of glucose is made by two low K
m hexokinases (230 and 64 M, respectively). As their level of activity shows significant changes over the moult cycle, these enzymes could be considered as having a regulatory role in the storage of glucose in the digestive gland.Abbreviations Acid Pase
aspecific acid phosphatase
- ATP
adenosine triphosphate
- DTT
dithiothreitol
- EDTA
ethylenediaminetetra-acetate
- G
calcium phosphate granules fraction
- G6P
glucose-6-phosphate
- G6Pase
hepatic glucose-6-phosphatase
- G6PDH
glucose-6-phosphate dehydrogenase
-
K
m
Michaelis-Menten constant
- MI
mitochondria and intermediate postmitochondrial particles
- N
nuclei fraction
- NADH
nicotineamide adenine dinucleotide
- P
microsome fraction
- Pi
inorganic phosphate
- PMSF
phenylmethylsulphonylfluoride
- STI
soybean trypsin inhibitor
- glyP
Na--glycerophosphate
- T1,2,3
transport protein 1,2,3
- TCA
trichloroacetic acid 相似文献
16.
Vladimir N. Podust Tamara O. Korobeinicheva George A. Nevinsky Asja S. Levina Olga I. Lavrik 《Molecular biology reports》1990,14(4):247-249
Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the Kd values for oligonucleotide primers of different length. The Kd values are smaller by a factor of 2.5 than the Km values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The Kd and Km values are nearly the same for Klenow fragment. Such approach to the determination of Km/Kd ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP
adenosine 2,3-riboepoxide 5-triphosphate
- KF
Klenow fragment of E. coli DNA polymerase I
- Pol I
E. coli DNA polymerase I
- Pol
human placenta DNA polymerase 相似文献
17.
Mohamed TM 《Experimental & applied acarology》2001,25(3):231-244
Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl2, and inhibited by CaCl2, MgCl2, NiCl2, and ZnCl2. 相似文献
18.
Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal4-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal4-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP
arabinogalactan-protein
- BSA
bovine serum albumin
- ELISA
enzyme-linked immunosorbent assay
- FITC
fluorescein isothiocyanate
- Gal3-BSA
-(16)-galactotriose coupled to BSA
- Gal4-BSA
-(16)-galactotetraose coupled to BSA
- Ig
immunoglobulin
- 4-Me-GlcpA
4-O-methyl-d-glucopyranosyluronic acid
- Mr
relative molecular mass
The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts. 相似文献
19.
Toxicity of 14 selected acaricides was tested for a laboratory strain of femaleD. gallinae. The most toxic were carbaryl (LC50=5.0 g/m2), deltamethrin (LC50=7.8 g/m2), bendiocarb (LC50=11.1g/m2), and permethrin (LC50=12.6 g/m2). High level resistance to DDT inD. gallinae was recorded, with factors of resistance at LC50/ LC90 reaching 57/73. 相似文献
20.
Purification and properties of gammagamma-enolase from pig brain 总被引:1,自引:0,他引:1
Isoelectric focusing revealed three enolase isoforms in pig brain, which were designated as - (pI = 6.5), - (pI = 5.6), and -enolase (pI = 5.2). The pI of purified -enolase was also 5.2. The -enolase isoform of enolase was purified from pig brain by a purification protocol involving heating to 55°C for 3 min, acetone precipitation, ammonium sulfate precipitation (40%–80%), DEAE Sephadex ion-exchange chromatography (pH 6.2), and Sephadex G200 gel filtration. The final specific activity was 82 units/mg protein. As with other vertebrate enolases, -enolase from pig proved to be a dimer with a native mass of 85 kDa and a subunit mass of 45 kDa. The pH optimum for the reaction in the glycolytic direction is 7.2. The K
m values for 2-PGA, PEP, and Mg2+ were determined to be 0.05, 0.25, and 0.50 mM, respectively, similar to K
m values of other vertebrate enolases. The amino acid composition of pig -enolase, as determined by amino acid analysis, shows strong similarity to the compositions of -enolases from rat, human, and mouse, as determined from their amino acid sequences. Despite the differences seen with some residues, and considering the ways that the compositions were obtained, it is assumed that pig -enolase is more similar than the composition data would indicate. Moreover, it is likely that the sequences of pig -enolase and the other -enolases are almost identical. Li+ proved to be a noncompetitive inhibitor with either 2-PGA or Mg2+ as the variable substrate. This enolase crystallized in the monoclinic space group P2, or P21. An R
symm <5% was obtained for data between 50 and 3.65 Å, but was a disappointing 30% for data between 3.65 and 3.10 Å, indicating crystal disorder. 相似文献