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1.
Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K
m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K
m of 0.033 mM), HGPRT (K
m of 0.074 mM), and PRPP amidotransferase (K
m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702. 相似文献
2.
Summary The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37–40° C, the optimum pH around 8.0 and optimal activity required 50–100 mM TRIS-HCI buffer, pH 8.0 and 3–7 mM MgCl2; MgCl2 in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occured only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The K
m values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the V
max values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a K
m of 0.17 mM and a V
max of 1.05 U/mg and for ATP a K
m of 0.16 mM and a V
max of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (K
i) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.
Correspondence to: J. Winter 相似文献
3.
Marta E. Farías Ana M. Strasser de Saad Aída A. Pesce de Ruiz Holgado Dr. Guillermo Oliver 《Current microbiology》1991,22(4):205-211
l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM
l-cysteine in 50 mM phosphate buffer. Mr=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (Mr=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent Km forl-serine was 65 mM. Fe++ was required for the enzymatic activity, and the apparent Km value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit. 相似文献
4.
Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through
the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.660 3.3) were shaken in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly.
However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium
solution or in the buffer containing 20 mM MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation
reaction by the resting cells.
Received: 28 June 2001 / Accepted: 30 July 2001 相似文献
5.
Choline is a necessary substrate of the lipid membrane and for acetylcholine synthesis. Accumulating evidence indicates that
besides being a structural component, choline is also a functional modulator of the membrane. It has been shown to be a muscarinic
acetylcholine receptor (mAChR) agonist and can induce a novel K+ current in cardiac cells. However, the potential role of choline in modulating cardiac functions remained unstudied despite
that mAChRs are known to be important in regulating heart functions. With microelectrode techniques, we found that choline
produced concentration-dependent (0.1∼10 mm) decreases in sinus rhythm and action potential duration in isolated guinea pig atria. The effects were reversed by 2 nm 4DAMP (an M3-selective antagonist). Whole-cell patch-clamp recordings in dispersed myocytes from guinea pig and canine atria revealed
that choline is able to induce a K+ current with delayed rectifying properties. The choline-induced current was suppressed by low concentrations of 4DAMP (2∼10
nm). Antagonists toward other subtypes (M1, M2 or M4) all failed to alter the current. The affinity of choline (K
d
) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 0.9 mm, consistent with the concentration needed for the current induction and for the HR and APD modulation. Our data indicate
that choline modulates the cellular electrical properties of the hearts, likely by activating a K+ current via stimulation of M3 receptors.
Received: 1 December 1998/Revised: 12 February 1999 相似文献
6.
NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of
Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively
charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of
NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments.
We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate
for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM KCl to 3 M KCl), (2) anion over cation selectivity (V
reversal = +16 mV in 250 mM KCl || 1 M KCl), and (3) two forms of voltage-dependent gating (channel closures above ±200 mV). Single-channel conductance decreases
by 50% when HEPES concentration is increased from 100 μM to 100 mM in 250 mM KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers,
we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate
concentration is increased from 0 mM to 5 mM in 250 mM KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes
are on the far side of agar–KCl bridges. A suitable baseline solution for NanC is 250 mM KCl adjusted to pH 7.0 without buffer. 相似文献
7.
A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His6-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K
m and V
max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10−3 mM min−1, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM
L-Gln, 40 mM ethylamine, and 1.04 U His6-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained. 相似文献
8.
The glucose 1-phosphate uridylyltransferase (GalU) is absolutely required for the biosynthesis of capsular polysaccharide,
the sine qua non virulence factor of Streptococcus pneumoniae. The pneumococcal GalU protein was overexpressed in Escherichia coli, and purified. GalU showed a pI of 4.23, and catalyzed the reversible formation of UDP-glucose and pyrophosphate from UTP
and glucose 1-phosphate with Km values of 0.4 mM for UDP-glucose, 0.26 mM for pyrophosphate, 0.19 mM for glucose 1-phosphate, and 0.24 mM for UTP. GalU has an optimum pH of 8–8.5, and requires Mg2+ for activity. Neither ADP-glucose nor TDP-glucose is utilized as substrates in vitro. The purification of GalU represents
a fundamental step to provide insights on drug design to control the biosynthesis of the main pneumococcal virulence factor. 相似文献
9.
Franklyn G. Healy Charles Latorre Stephan L. Albrecht Pallavolu M. Reddy Keelnatham T. Shanmugam 《Current microbiology》2003,46(6):0423-0431
A L-methionine-D,L-sulfoximine-resistant mutant of the cyanobacterium Anabaena variabilis, strain SA1, excreted the ammonium ion generated from N2 reduction. In order to determine the biochemical basis for the NH4
+-excretion phenotype, glutamine synthetase (GS) was purified from both the parent strain SA0 and from the mutant. GS from
strain SA0 (SA0-GS) had a pH optimum of 7.5, while the pH optimum for GS from strain SA1 (SA1-GS) was 6.8. SA1-GS required
Mn+2 for optimum activity, while SA0-GS was Mg+2 dependent. SA0-GS had the following apparent K
m
values at pH 7.5: glutamate, 1.7 mM; NH4
+, 0.015 mM; ATP, 0.13 mM. The apparent K
m
for substrates was significantly higher for SA1-GS at its optimum pH (glutamate, 9.2 mM; NH4
+, 12.4 mM; ATP, 0.17 mM). The amino acids alanine, aspartate, cystine, glycine, and serine inhibited SA1-GS less severely than the SA0-GS. The nucleotide
sequences of glnA (encoding glutamine synthetase) from strains SA0 and SA1 were identical except for a single nucleotide substitution that
resulted in a Y183C mutation in SA1-GS. The kinetic properties of SA1-GS isolated from E. coli or Klebsiella oxytoca glnA mutants carrying the A. variabilis SA1 glnA gene were also similar to SA1-GS isolated from A. variabilis strain SA1. These results show that the NH4
+-excretion phenotype of A. variabilis strain SA1 is a direct consequence of structural changes in SA1-GS induced by the Y183C mutation, which elevated the K
m
values for NH4
+ and glutamate, and thus limited the assimilation of NH4
+ generated by N2 reduction. These properties and the altered divalent cation-mediated stability of A. variabilis SA1-GS demonstrate the importance of Y183 for NH4
+ binding and metal ion coordination.
Received: 3 July 2002 / Accepted: 29 July 2002 相似文献
10.
N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co2+ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co2+ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn2+ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h. 相似文献
11.
Nobuyuki Uozumi Yoshihiro Kato Yutaka Nakashimada Takeshi Kobayashi 《Applied microbiology and biotechnology》1992,37(5):560-565
Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl2 or MgCl2 was found to be the most effective agent for excretion among other combinations. CaCl2 supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl2 addition during the culture. Repeated batch culture with 50 mM CaCl2 supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl2 supplementation.
Correspondence to: T. Kobayashi 相似文献
12.
G. Vengadesan N. Selvaraj R. Prem Anand V. Gaba A. Ganapathi 《In vitro cellular & developmental biology. Plant》2005,41(6):789-793
Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion.
Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid
medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM
l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of
heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium
containing 175.2 mM sucrose and 0.5 gl−1 activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6
mM sucrose and 1.4 μM gibberellic acid (GA3) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants. 相似文献
13.
Summary Free-living or immobilized Chlamydomonas reinhardtii cells photoproduce ammonium from nitrite in a medium containing 1 mM of l-methionine-d,l-sulphoximine (MSX). Ammonium is accumulated in the medium to 8 mM final concentration, which inhibits nitrite uptake by the MSX-treated cells and consequently the excretion of ammonium is blocked. However, if ammonium was removed from the medium and nitrite and MSX periodically restored, the photoproduction process could be maintained over 96 h, with a final ammonium concentration of about 18 mM for free-living cells and 28 mM for immobilized ones. The MSX-treated cells showed a photoproduction productivity of 1300 mol NH
4
+
· mg chlorophyll (Chl)-1, with an average production rate of 14 mol NH
4
+
· mg Chl-1 per hour, for calcium alginate-entrapped cells, while the corresponding data for free-living ones was 650 mol NH
4
+
· mg Chl-1 and 6.7 mol NH
4
+
· mg Chl-1 per hour, respectively. Immobilized cells showed a significant increase in the nitrite uptake rate, probably due to a change in membrane permeability as a consequence of cell-matrix interactions. 相似文献
14.
Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (Mr) of 600 000 is composed of monomeric sub-units of 60 000 (Mr). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K
m values (K
m
app
) in the biosynthetic reaction for glutamate, NH4Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K
m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF
phenylmethylsulfonylfluoride
- TCA
trichloroacetic acid
- GS
glutamine synthetase
- MSO
L-Methionine-D-L-sulfoximine
- SDS-PAGE
sodium dodecyl sulfatepolyacrylamide gel electrophoresis
- SVPDE
snake venum phosphodiesterase 相似文献
15.
Sigrun Feldmann Georg A. Sprenger Hermann Sahm 《Applied microbiology and biotechnology》1989,31(2):152-157
Summary Grown anaerobically on d-xylose, Klebsiella planticola ATCC 33531 produced acetate, formate, lactate, CO2 and ethanol as major end-products. A Mu-insertion mutant which lacked pyruvate-formate-lyase showed among its fermentation products more than 70% d-lactate with residual acetate, 2,3-butanediol, and traces of ethanol, formate, and CO2. After the introduction of a plasmid carrying the gene for the enzyme pyruvate decarboxylase from Zymomonas mobilis, this Klebsiella mutant became an efficient ethanol producer. The recombinant strain produced 387 mM ethanol from 275 mM xylose in 80 h, about 83% of the theoretical maximal yield. Furthermore, this mutant consumed more than double the amount of xylose (41 g/l) compared to the wild-type, due to reduced production of inhibiting acids during growth.Dedicated to Professor Dr. Zähner on the occasion of his 60th birthday 相似文献
16.
The effect of transnitrosation intermediate between S-nitroso-N-acetylcysteine (NACysNO) and cysteine on the growth of vgb-bearing Enterobacter aerogenes was investigated using three parameters: the ratio of the specific growth rates, the inhibition zone, and α-amylase synthesis for the culture exposed to stressors
to that of the same stressor-free cultures. The effect of NACysNO/cysteine on the growth of Enterobacter strains was distinctive as compared with the CysNO, NACysNO, and their combination. At a higher concentration (2 mM), the extents of inhibition based on the μNACysNO/cysteine/μno stress ratio for these cultures were 57%, 62%, and 68% for VHb-expressing, parental, and pUC9-harboring cells, respectively. The
inhibition caused by 2 mM NACysNO in the presence of 1 mM cysteine in all bacterial strains was almost twofold that achieved by NACysNO alone. Based on the diameter of the inhibition
zone and α-amylase productivity, the four compounds (NACysNO/Cysteine, CysNO, NACysNO, and their combinations) affected the
E. aerogenes strains in a concentration-dependent and negative manner. This negative effect was lower in vgb-bearing than vgb-lacking strains. Thus, sulfur-to-sulfur transnitrosation was an efficient NO release and significantly (P < 0.05) affects the growth of Enterobacter strains, to a lesser extent in vgb-bearing strains. 相似文献
17.
M. Iino S. Ciani K. Tsuzuki S. Ozawa Y. Kidokoro 《The Journal of membrane biology》1997,155(2):143-156
Ion permeation properties of the mouse e2/ζ1 NMDA receptor channel expressed in Xenopus oocytes were studied using the outside-out patch-clamp technique. In symmetrical Na+ solutions, the single-channel I-V relations were almost linear at low electrolyte concentrations, but rectified inwardly for Na+ concentrations above 50 mm. In symmetrical Na+ solutions, the ``zero-current conductance' increased with Na+ concentration and saturated according to a hyperbolic curve, the half-maximal saturating activity, K
M
(Na), being 14.2 mm and the maximal conductance, G
max(Na), 53.9 pS. When Ca2+ was present with Na+ in the external solution, the single-channel current was lower than in pure Na+, although the reversal potential indicated a higher permeability for Ca2+ than for Na+. Using ion activities, PCa/PNa was found to be about 17. The I-V data were fitted with a model based on the Eyring's rate theory, assuming a one-ion pore with three energy barriers and two
sites. The K
M
(Ca) and G
max (Ca) were 76.5 μm and 21.2 pS, respectively. According to the estimated rate constants, K
M
for Ca2+ is mainly determined by the binding strength of a site located 80% away from the channel opening at the external membrane-solution
interface, a position similar to that postulated previously for the Mg2+ blocking site.
Received: 3 May 1996/Revised: 25 September 1996 相似文献
18.
P. Kugler 《Histochemistry and cell biology》1990,93(5):537-540
Summary An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat
sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM
l-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. — The study showed that in the histochemical demonstration of Gldh the use of relatively
high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial
matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable
Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was
not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium
ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences
in demonstrable activities.
Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2) 相似文献
19.
Rebecca C. Garland Carl F. Cori Hsia-Fei W. Chang 《Molecular and cellular biochemistry》1976,12(1):23-31
Summary Iodoacetamide, N-ethylmaleimide, p-hydroxy-mercuribenzoate (p-MB) and HgCl2 were tested as inhibitors of microsomal glucose-6-phosphatase. Iodoacetamide had no effect at 2mm. N-ethylmaleimide inhibited only crude, but not purified microsomal preparations (M2) or crude microsomes exposed to deoxycholate.14C-labelled N-ethylmaleimide was not bound by the M2 protein fraction. p-MB inhibited all types of preparations and the inhibition was not counteracted by detergent. A more detailed study was carried out with the purified M2 fraction (specific activity: 2–4µmoles Pi/min/mg protein). Glucose-6-phosphate hydrolysis was inhibited 50% by 5 × 10–5
m p-MB. The inhibition was completely reversible by dithiothreitol except when the enzyme was pre-incubated with p-MB in the absence of substrate. Then p-MB accelerated the temperature-dependent inactivation of glucose-6-phosphatase. Binding studies showed that around 3µmoles14C-p-MB were incorporated into 100 mg M2 protein regardless of the concentration of mercurial in the incubation mixture. That is, over a 25 fold range of p-MB concentration, causing up to 80% inhibition of enzyme activity, no difference was seen in the amount of labelled p-MB which was irreversibly bound to M2 protein. Kinetically p-MB behaved like a reversible inhibitor and this was confirmed by dilution experiments. Several compounds, including some amino acids, antagonized the inhibition by p-MB. The order of effectiveness was EDTA > barbital > tryptophan > histidine > lysine > other amino acids. Glycine, Tris and urea were ineffective competitors of p-MB inhibition. Double reciprocal plots showed that the Km for glucose-6-phosphate was increased and the Vmax reduced in the presence of p-MB. HgCl2 was a more effective inhibitor than p-MB with a Ki of 6 × 10–6
m. We conclude that a reaction of p-MB with M2 sulfhydryls does not play a part in the inhibition of enzyme activity. It is suggested that p-MB may interact with one or more amino acid side chains in such a way that enzyme conformation is altered.Supported by U.S. Public Health Service Grant No. AM11448-08 and General Research Support Grant No. RR05486-12. 相似文献
20.
Summary This study was undertaken in order to determine directly the rates of K leakage (J
K) out of the tips of microelectrodes into a solution of 100 mM KCl (approximating the K concentration of the cell interior) and to relate these rates to the concentration of the filling solution and the tip resistance. The values ofJ
K for electrodes filled with 3m KCl having resistances of 16 and 30 M (when measured in 3m KCl) were 10 and 5.5 fmol/sec, respectively. When the same electrodes were filled with 0.5m KCl, the resistances (measured in 0.5m KCl) increased to 62 and 115 M, respectively, andJ
K fell to 1.8 and 1.0 fmol/sec, respectively. These values are in reasonable agreement with what would be expected from theoretical considerations if leakage of KCl were the result of diffusion plus convective flow due to the hydrostatic pressure of the filling solution.We conclude that K leakage out of microelectrodes filled with 3m KCl is unnecessarily high; leakage can be reduced fivefold by filling electrodes with 0.5m KCl without incurring significant increases in tip or diffusion potentials or unmanageable tip resistances.Finally, the lowest rate of K leakage observed (1 fmol/sec) is still very considerable for the case of animal cells with an intracellular volume of approximately 1 pl and a K content of approximately 100 fmol. The finding of stable intracellular potentials, often for many minutes, in some tissues suggests that K which enters the cell rapidly diffuses into neighboring cells via high conductance intercellular communications. 相似文献