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1.
Two lactate dehydrogenase (LDH) mutations were recovered independently among offspring of ethylnitrosourea-treated male mice by screening for alterations of isoelectric focusing pattern in liver homogenates. Investigations of physicochemical and kinetic properties of the mutant enzymes indicated that the mutant traits resulted from point mutations at the Ldh-1 structural locus. Therefore, the new alleles were designated Ldh-1
a-m5Neu and Ldh-1
a-m6Neu, respectively. Both mutant alleles code for proteins which exhibit an altered stability to heat, in addition to changes in isolectric focusing pattern and a reduction in anodal electrophoretic mobility. While LDH-A a-m5Neu proteins are markedly less heat stable, LDH-A a-m6Neu proteins are more heat stable than the wild-type enzyme. Furthermore, a small elevation of K
m for pyruvate, a slightly reduced inhibition by high pyruvate concentrations, and a slight acidic shift of the pH activity profile distinguish LDH-A a-m6Neu from both wild-type and LDH-A a-m5Neu enzymes. Significant alterations of LDH activity were detected in some tissues from LDH-A a-m5Neu individuals but not in those from LDH-A a-m6Neu animals. Erythrocytes and blood of LDH-A a-m5Neu mutants revealed activity levels which were reduced by approximately 6 and 13% compared with those of wild types in heterozygous and homozygous individuals, respectively. In addition, an elevation of approximately 6% in LDH activity was found in skeletal muscle in homozygous mutants. Consistent with the unaltered or only slightly altered LDH activity in tissues, the genetic as well as the physiological characterization yielded no easily detectable effects from either mutation on metabolism or fitness of the affected individuals.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities. 相似文献
2.
The autosomal variation and the genetic control of GPI has been determined by a comparison of electrophoretic patterns of F 1 and backcross progeny of three inbred strains of mice. The locus controlling the production of GPI in the mouse has been designated Gpi-1. Two alleles at this locus have been described and designated Gpi-1
a and Gpi-1
b, which represent, respectively, the slow and fast electrophoretic forms. Twenty-seven inbred strains of mice have been classified for these two alleles. The absence of close linkage of Gpi-1 to seven other genetic loci has been determined. It has been demonstrated that the polymorphism of Gpi-1 is widely distributed in feral mice. GPI was expressed in vitro and in four types of malignant tumors.Supported by U.S. Public Health Service Grants GM-09966, from General Medical Sciences, and GY 4193. 相似文献
3.
Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in
whole blood have been recovered. The mutant line Ldh1
a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1
a7Neu, Ldh1
a11Neu, and Ldh1
a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the
number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical
and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1
a2Neu carried an A/T → G/C transition in codon 112 (in exon 3), resulting in an Asn → Asp substitution; Asn112 is part of the helix
αD, which is involved in the coenzyme-binding domain. Ldh1
a7Neu contained an A/T → C/G transversion within the codon for residue 194 in exon 4, causing an Asp → Ala substitution, which
may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1
a11Neu in exon 7: the transition C/G → T/A, a silent mutation, and two transversions C/G → A/T and C/G → G/C, both missense mutations,
which led to the amino acid replacements Ala319 → Glu and Thr321 → Ser, respectively, located in the αH helix structure of
the COOH tail of LDHA. We suggest that the mutation is the result of a gene conversion event between Ldh1
a wild-type gene and the pseudogene Ldh1-ps. The alteration Ile → Thr of codon 241 in exon 6 caused by the base pair change T/A → C/G was identified in the mutation
Ldh1
a12Neu; IIe241 is included in the helix α2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations
has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical
and physiological alterations.
Received: 3 July 1997 / Accepted: 30 September 1997 相似文献
4.
We have found a new allele at the structural locus for glucosephosphate isomerase (called Gpi-1
c
) in a population of wild mice. The Gpi-1
c
allele codes for an enzyme of greater cathodal electrophoretic mobility than either the Gpi-1
a
or Gpi-1
b
alleles found in the wild and in the SM/J and C57BL/6J inbred strains. Mice homozygous for Gpi-1
c
have erythrocyte enzyme activity reduced to 33% of normal levels, altered pH profile, lowered heat stability, and normal K
m
's when compared with SM/J and C57BL/6J mice. The activity of the enzyme in brain, liver, and kidney is not so markedly lowered, although the electrophoretic mobility, pH profile, and heat stability are altered in these tissues. Deficiencies of erythrocyte glucosephosphate isomerase in man, to this level, can cause severe hemolytic anemia. Homozygotes for Gpi-1
c
show only mild hematological symptoms. The frequency of Gpi-1
c
in wild populations of mice is discussed and the occurrence of a further rare allele Gpi-1
d
is reported.This work was supported by M.R.C. grants to Professor R. J. Berry and Dr. H. Kacser, whom we should also like to thank for much help and useful discussion. 相似文献
6.
Zymograms of glucosephosphate isomerase (GPI) and phosphogluconate dehydrogenase (PGD) revealed three isozymes for each enzyme in the plant species Scabiosa columbaria. Intergenic heterodimers are formed between the polypeptides coded by Gpi-1 and Gpi-2 and between those coded by Pgd-1 and Pgd-2, indicating that a GPI and a PGD locus have been duplicated in the past. The ancestral genes assort independently with their duplicated gene, suggesting that the duplications have originated from a process of translocation. Linkage was found only between Gpi-1 and Pgd-2 and between Gpi-2 and Pgd-1, suggesting that the duplicated loci were located on the same translocated chromosomal segment. Both duplications are present in all other examined species of Scabiosa and in Cephalaria and Knautia, two other genera of the Dipsacaceae. The genera Succisa and Dipsacus, also belonging to the Dipsacaceae, do not show Gpi-1 activity, making Gpi-2 and Pgd-1 the most likely ancestral genes. In Succisa, the isozymes of Gpi-1 and Gpi-2 either overlap or Gpi-1 has been silenced. The combined results suggest that a chromosomal segment containing Gpi-2 and Pgd-1 has been translocated before the divergence of Scabiosa, Cephalaria, Knautia, and Succisa. 相似文献
7.
The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln 3+ binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln 3+ binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln 3+ binding sites for Hb can be classified into three categories: i.e. positive cooperative sites ( NI), non-cooperative strong sites ( NS) and non-cooperative weak sites ( NW) with binding constants in decreasing order: KI> KS> KW. The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln 3+ concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln 3+ binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln 3+ concentration. At the range of [Ln 3+]/[Hb]<2, the marked increase of oxygen affinity ( P50 decrease) with the Ln 3+ concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln 3+ concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln 3+ binding. This was indicated by the changes in secondary structure characterized by the decrease of α-helix content. 相似文献
8.
Quantitative electrophoretic studies of the three allozymes of glucose phosphate isomerase (GPI-1) produced by Gpi-1s
a/Gpi-1s c heterozygous mice revealed two opposing influences on GPI-1 activity. First, the GPI-1 AC heterodimer is less stable than GPI-1 AA but more stable than the GPI-1 CC homodimer. Second, a genetic determinant that maps close to or within the Gpi-1s structural gene causes elevated activity of GPI-1 AC and probably also GPI-1 CC dimers. The relative lability of these allozymes masks this elevated activity in some tissues but the effect is probably ubiquitous. The significance of these observations is discussed.This study was begun while JDW was at the MRC Radiobiology Unit and continued at the Department of Obstetrics and Gynaecology of the University of Edinburgh, where it was supported, in part, by a grant from the Moray Endowment Fund. 相似文献
9.
Nitrate assimilation-defective mutants SP7, SP9, and SP17 of the cyanobacterium Anabaena sp. PCC 7120 were isolated by use of transposon mutagenesis and screened on medium containing chlorate. SP7 and SP17 represented
nitrate reductase-defective nature, while mutant SP9 appeared to be a regulatory mutant exhibiting pleiotropic behavior. Kinetics
of nitrate uptake system exhibited K
s values of 31–38 μM for parent, SP7, and SP17 strains; however, mutant SP9 exhibited a high K
s value of 109.5 μM. Defective nitrate reductase was apparent in mutant SP7 and SP9, while mutant SP17 exhibited partial defective
nature. Methyl viologen-dependent NR activity in parent strain presented a biphasic nature with K
m values of 0.13 and 2.47 mM, whereas a single K
m value (2.96 mM) was observed for mutant SP17. Mutant SP9 was also defective in nitrite uptake and reduction. Mutant strains
exhibited derepressed nitrogenase activity in the presence of nitrate, while glutamine synthetase activity remained unaltered.
Received: 20 April 1999 / Accepted: 22 May 1999 相似文献
10.
Summary As part of a genetic study of the mechanisms for cation transport in cultured mammalian cells, two mouse fibroblastic cell lines have been compared with respect to unidirectional 42K + influx. The cell lines are LM(TK
–) and LTK-5, a mutant selected from LM(TK
–) by the ability to grow in medium containing 0.2 mm K +. In both cell lines, the overall influx can be resolved into three components: (i) a ouabain- and vanadate-sensitive component (
i
M K
f), presumably the Na/K pump, which is a saturable function of extracellular K + with a K
1/2 of 1.3 mm; (ii) a furosemide-sensitive component (
i
M k
fx), also a saturable function of extracellular K +, with a K
1/2 of 6 mm; and (iii) a diffusional component (
i
M k
d); which is a linear function of extracellular K +.By several independent criteria,
i
M k
o and
i
M k
f appear to be distinct transport processes. First, as indicated above, they can be separated with the use of inhibitors. In addition, they can be separated genetically, since the LTK-5 mutant shows a threefold elevation in
i
M k
f with no change in
i
M k
o. And finally, extracellular Na + has no effect on
i
M k
o, but stimulates
i
M k
f, a result consistent with the notion that
i
M k
f influx occurs by Na–K cotransport.Further experiments were directed towards understanding the nature of the LTK-5 mutation and the physiological role of
i
M k
f. LTK-5 differs from the parental cell line, not only in having an increased
i
M k
f, but also in having a large cell volume, a slow maximal growth rate, and an ability to grow at 0.2 mm K +. The most straightforward interpretation — that the increased
i
M k
f is itself responsible—is unlikely since the addition of furosemide to the growth medium had no effect upon the growth rate or cell volume of the mutant at either normal or low extracellular K + concentrations. It did, however, render the parent capable of growth at 0.2 mm K +. Possible interpretations are discussed. 相似文献
11.
Anti-Pr cold agglutinins (CAs) with the subspecificities anti-Pr 1h,-Pr 1d, -Pr 2, -Pr 3h, -Pr 3d, -Pr M and anti-Sa CAs recognize immunodominant N-acetylneuraminic acid (Neu N Ac) groups of tetra and/or trisaccharides (O-glycans) of glycophorin. These O-glycans are sialylated in 2,3- and/or 2,6-linkages. Sa and most Pr antigens have been inactivated by 2,3-specific sialidases. Antigenicity was reconstituted on desialylated glycophorin by 2,3-specific Gal1,3Gal N Ac-sialyltransferase indicating that 2,3-linked Neu N Ac groups are the immunodominant components of Sa and most Pr antigens. Some Pr antigens were resistant to 2,3-specific sialidase and were not reconstituted by 2,3-specific Gal1,3Gal N Ac-sialyltransferase, which indicates that 2,6-linked Neu N Ac group represents an immunodominant component of some Pr antigens. 相似文献
12.
The Arabidopsis thaliana cDNA, KAT1 encodes a hyperpolarization-activated K + (K +
in
) channel. In the present study, we identify and characterize dominant negative point mutations that suppress K +
in
channel function. Effects of two mutations located in the H5 region of KAT1, at positions 256 (T256R) and 262 (G262K), were
studied. The co-expression of either T256R or G262K mutants with KAT1 produced an inhibition of K + currents upon membrane hyperpolarization. The magnitude of this inhibition was dependent upon the molar ratio of cRNA for
wild-type to mutant channel subunits injected. Inhibition of KAT1 currents by the co-expression of T256R or G262K did not
greatly affect the ion selectivity of residual currents for Rb +, Na +, Li +, or Cs +. When T256R or G262K were co-expressed with a different K + channel, AKT2, an inhibition of the channel currents was also observed. Voltage-dependent Cs + block experiments with co-expressed wild type, KAT1 and AKT2, channels further indicated that KAT1 and AKT2 formed heteromultimers.
These data show that AKT2 and KAT1 are able to co-assemble and suggest that suppression of channel function can be pursued
in vivo by the expression of the dominant negative K
+
in
channel mutants described here.
Received: 2 July 1998/Revised: 23 October 1998 相似文献
13.
ObjectivesThis study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications. ResultsSite-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (kcat/Km: 44.14 s?1 mM?1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL. ConclusionsOur study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry. 相似文献
14.
Single mutants (C62S, C62V, C86S, C146S, C164S), double mutants (C62/146S, C62/164S, C86/146S, C146/164S), and triple mutant
C62/146/164S of the Luciola mingrelica firefly luciferase carrying C-terminal His6-tag were obtained on the basis of plasmid pETL7 by site-directed mutagenesis.
Bioluminescence and fluorescence spectra were not altered by the introduced mutations. In the case of mutants C86S, C86/146S,
C62/164S, and the triple mutant C62/146/164S, the K
mATP and KmLH2 K_m^{LH_2 } values were increased by a factor of ∼1.5–1.9. Their expression level, specific activity, and thermal stability were significantly
decreased. The other mutations had almost no effect on the K
mATP and KmLH2 K_m^{LH_2 } values, specific activity, and thermal stability of the enzyme. Thermal stability of the C146S mutant was increased by a
factor of ∼2 and 1.3 at 37 and 42°C, respectively. The possible mechanism of the influence of these mutations on properties
and structure of the enzyme is discussed. 相似文献
15.
A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K + or Rb +, but not by Na +, over the range of zero to 100mM was shown in the isolated membranes of E. coli grown anaerobically in the presence of glucose. This effect was observed only in parent and in the trkG, but not in the trkA, trkE, or trkH mutants. The trkG or the trkH mutant with an unc deletion had a residual ATPase activity not sensitive to DCCD. A stimulation of the DCCD-sensitive ATPase activity by K + was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate. Growth of the trkG, but not of other trk mutants, in the medium with moderate K + activity did not depend on K + concentration. Under upshock, K + accumulation was essentially higher in the trkG mutant than in the other trk mutant. The K +-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grown E. coli has been shown to depend absolutely on both the F 0F 1 and the Trk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H +-K + pump. The trkG gene is most probably not functional in anaerobically grown bacteria.This study was performed at the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637. 相似文献
16.
Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-aminocephalosporanic acid (7-ACA). Currently, in the pharmaceutical industrie, 7-ACA is mainly produced from cephalosporin C by sequential application of D -amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme γ-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type γ-glutamyltranspeptidase of B. subtilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a kcat value of 0.0485 s -1. Its activity has been enhanced by site directed and random mutagenesis. The kcat/ Km value was increased to 3.41 s -1 mM -1 for a E423Y/E442Q/D445N mutant enzyme and the kcat value was increased to 0.508 s -1 for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000-fold and 10-fold, compared with the wildtype γ-glutamyltranspeptidase of B. subtilis. 相似文献
17.
2,3-Diphosphoglycerate (2,3-DPG), an intracellular metabolite of glycolytic pathway is known to affect the oxygen binding capacity of haemoglobin and mechanical properties of the red blood cells. 2,3-DPG levels have been reported to be elevated during anaemic conditions including visceral leishmaniasis. 2,3-DPG activity in P. falciparum infected red blood cells, particularly in cells infected with different stages of the parasite and its relationship with structural integrity of the cells is not known. Chloroquine sensitive and resistant strains of P. falciparum were cultured in vitro and synchronized cultures of ring, trophozoite and schizont stage rich cells along with the uninfected control erythrocytes were assayed for 2,3-DPG activity and osmotic fragility. It was observed that in both the strains, in infected erythrocytes the 2,3-DPG activity gradually decreased and osmotic fragility gradually increased as the parasite matured from ring to schizont stage. The decrease in 2,3-DPG may probably be due to increased pyruvate kinase activity of parasite origin, which has been shown in erythrocytes infected with several species of Plasmodium. The absence of compensatory increase in 2,3-DPG in P. falciparum infected erythrocytes may aggravate hypoxia due to anaemia in malaria and probably may contribute to hypoxia in cerebral malaria. As 2,3-DPG was not found to be increased in erythrocytes parasitized with P. falciparum, the increased osmotic fragility observed in these cells is not due to increased 2,3-DPG as has been suggested in visceral leishmaniasis. 相似文献
18.
Commercial cultivation of Spirulina sp. is highly popular due to the presence of high amount of C‐phycocyanin (C‐PC ) and other valuable chemicals like carotenoids and γ‐linolenic acid. In this study, the pH and the concentrations of nitrogen and carbon source were manipulated to achieve improved cell growth and C‐PC production in NaCl‐tolerant mutant of Spirulina platensis . In this study, highest C‐PC (147 mg · L ?1) and biomass (2.83 g · L ?1) production was achieved when a NaCl‐tolerant mutant of S. platensis was cultivated in a nitrate and bicarbonate sufficient medium (40 and 60 mM, respectively) at pH 9.0 under phototrophic conditions. Kinetic study of wildtype S. platensis and its NaCl‐tolerant mutant was also done to determine optimum nitrate concentrations for maximum growth and C‐PC production. Kinetic parameter of inhibition (Haldane model) was fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that the maximum specific growth rate (μ max) for NaCl‐tolerant mutant increased by 17.94% as compared to its wildtype counterpart, with a slight increase in half‐saturation constant (K s), indicating that this strain could grow well at high concentration of NaNO 3. C‐PC production rate (C max) in mutant cells increased by 12.2% at almost half the value of K s as compared to its wildtype counterpart. Moreover, the inhibition constant (K i) value was 207.85% higher in NaCl‐tolerant mutant as compared to its wildtype strain, suggesting its ability to produce C‐PC even at high concentrations of NaNO 3. 相似文献
19.
Responses of leaf and shoot hydraulic conductance to light quality were examined on shoots of silver birch ( Betula pendula), cut from lower (‘shade position’) and upper thirds of the crowns (‘sun position’) of trees growing in a natural temperate forest stand. Hydraulic conductances of leaf blades ( Klb), petioles ( KP) and branches (i.e. leafless stem; KB) were determined using a high pressure flow meter in steady state mode. The shoots were exposed to photosynthetic photon flux density of 200–250 µmol m ?2 s ?1 using white, blue or red light. Klb depended significantly on both light quality and canopy position ( P < 0.001), KB on canopy position ( P < 0.001) and exposure time ( P = 0.014), and none of the three factors had effect on KP. The highest values of Klb were recorded under the blue light (3.63 and 3.13 × 10 ?4 kg m ?2 MPa ?1 s ?1 for the sun and shade leaves, respectively), intermediate values under white light (3.37 and 2.46 × 10 ?4 kg m ?2 MPa ?1 s ?1, respectively) and lowest values under red light (2.83 and 2.02 × 10 ?4 kg m ?2 MPa ?1 s ?1, respectively). Light quality has an important impact on leaf hydraulic properties, independently of light intensity or of total light energy, and the specific light receptors involved in this response require identification. Given that natural canopy shade depletes blue and red light, Klb may be decreased both by reduced fluence and shifts in light spectra, indicating the need for studies of the natural heterogeneity of Klb within and under canopies, and its impacts on gas exchange. 相似文献
20.
Summary A group of chlorophyll deficient mutants ( br
s mutants) of Chlamydomonas accumulates protoporphyrin and has poorly developed chloroplast membrane systems (Wang et al. 1974). In order to determine whether a poorly developed chloroplast membrane system is the reason for, or the result of, the inability of the br
s mutants to metabolize protoporphyrin to chlorophyll, a second mutation was selected which restored chlorophyll synthesis in br
s mutants. One such double mutant ( br
s- 2 g-4) was analyzed. The double mutant br
s- 2 g-4 has partially restored chlorophyll synthesis, but has defective photosystem II and photosystem I electron transport as well as abnormal chloroplast ultrastructure. Since these defects are not present in cells carrying only the g-4 mutation, they are presumed to be caused by the br
s- 2 mutation. It is concluded that a defect in chloroplast membrane development resulting from the br
s- 2 mutation causes an apparent defect in magnesium chelation by protoprophyrin. This is consistant with evidence that chlorophyll biosynthesis from magnesium protoporphyrin to chlorophyll takes place on the chloroplast membranes. 相似文献
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