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1.
Maneerat Koohapitagtam Suang Rungpragayphan Ratchanee Hongprayoon Wichai Kositratana Theerapol Sirinarumitr 《Molecular biology reports》2010,37(4):1677-1683
Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As
a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers.
PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss
of antibody diversities. To overcome this problem, we described a novel two-step amplification of Vκ and VH genes with newly designed primer sets. Initially, we amplified Vκ and VH genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly
degenerated primers were used to amplify the Vκ and VH genes from the framework region 1 to the joining region. The Vκ and VH genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from
the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability
of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of
this strategy and the primer sets for construction of the scFv library. 相似文献
2.
Benzoylecgonine is a major metabolite of cocaine. We generated hybridoma cells (C1303) producing anti-benzoylecgonine monoclonal
antibody (mAb) with a single-chain variable fragment (scFv) and an antigen-binding domain from the C1303 cells. Genes encoding
an scFv antibody and constant region (Fc) were amplified from a cDNA library of C1303 cells using PCR. The two frameworks
built for scFv and scFv-Fc consisted of HL [(heavy chain variable region, VH) — linker — (light chain variable region, VL)] and HL-Fc, respectively. A 45 base-pair-long sequence encoding (Gly4-Ser)3 was used as the linker, and the mouse IgG1 constant region sequence (225 amino acids) was used as the Fc domain. These two
types of recombinant Abs were determined to be 750 bp in length (which corresponds to a 30 kDa protein) in the HL and 1,432
bp in length (which corresponds to a 65 kDa protein) in the HL-Fc, respectively. The parental Ab and HL-Fc affinities against
benzoylecgonine were measured by ELISA and found to be nearly equal to the Ab concentration. We were also able to measure
HL affinity using an agarose diffusion assay (Ouchterlony test). The affinity of the recombinant single-chain antibody against
benzoylecgonine was sufficiently comparable to that of the parent antibodies to be used for the immunodetection of specific
drug compounds or the detoxification of drug abusers by immunotherapy. 相似文献
3.
4.
Druar C Saini SS Cossitt MA Yu F Qiu X Geisbert TW Jones S Jahrling PB Stewart DI Wiersma EJ 《Immunogenetics》2005,57(10):730-738
The cynomolgus macaque, Macaca fascicularis, is frequently used in immunological and other biomedical research as a model for man; understanding it's antibody repertoire
is, therefore, of fundamental interest. The expressed variable-region gene repertoire of a single M. fascicularis, which was immune to the Ebola virus, was studied. Using 5′ rapid amplification of cDNA ends with immunoglobulin (Ig)G-specific
primers, we obtained 30 clones encoding full-length variable, diversity, and joining domains. Similar to the human VH repertoire, the M. fascicularis repertoire utilized numerous immunoglobulin heavy variable (IGHV) gene fragments, with the VH3 (41%), VH4 (39%), and VH1 (14%) subgroups used more frequently than the VH5 (3.9%) or VH7 (1.7%) subgroups. Diverse immunoglobulin heavy joining (IGHJ) fragments also appeared to be utilized, including a putative
homolog of JH5β gene segment identified in the related species Macaca mulatta, Rhesus macaque, but not in humans. Although the diverse V region genes in the IgG antibody repertoire of M. fascicularis had likely undergone somatic hypermutations (SHMs), they nevertheless showed high nucleotide identity with the corresponding
human germline genes, 80–89% for IGHV and 72–92% for IGHJ. M. fascicularis and human VH genes were also similar in other aspects: length of complementarity-determining regions and framework regions, and distribution
of consensus sites for SHMs. Finally, we demonstrated that monoclonal antibodies (mAbs) specific for an Ebola protein could
be obtained from M. fascicularis tissue samples by phage display technology. In summary, the study provides new insight into the M. fascicularis V region gene repertoire and further supports the idea that macaque-derived mAbs may be of therapeutic value to humans. 相似文献
5.
Cohen SD 《Microbial ecology》2006,52(3):463-469
Discula umbrinella, a fungal endophyte of oak species, colonizes and reproduces on leaves of Quercus alba and Q. rubra in forest ecosystems. Twenty-nine isolates collected from leaves of both oak species (16 from Q. alba and 13 from Q. rubra) were assayed for oak species preference and genetic variation based on primer-specific polymerase chain reactions for the intergenic spacer region (IGS) of ribosomal DNA. DNA sequencing of the polymerase chain reaction products revealed a 10-bp insertion (237–247 bp) at the 3′ end of the IGS region present in nine isolates and absent in 20 of the isolates. Phylogenetic analysis of the IGS region using the neighbor-joining method identified IGS groups (groups I–V) based on single nucleotide sequence differences. Host selectivity and geographic origin of isolates were correlated in some instances with the IGS groups. Isolates within each IGS group were further analyzed for nucleotide polymorphisms to confirm genotype identity and genotype diversity. Ten different genotypes (Va–Vj) were identified among the isolates analyzed. Genotype diversity was greatest in IGS groups I, IV, and V. Seventy percent of the genotypes (Vc, Vd, Ve, Vf, Vg,Vi, and Vj) contained isolates with single tree species preferences. 相似文献
6.
Hoh JF Withers KW Zhong WW 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2008,178(3):279-284
The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms
and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin
components were found: V1a, V1b,V1c, V2 and V3, in order of decreasing mobility. In western blots, V1a, V1b, and V1c reacted with anti-α-MyHC antibody, but not with anti-β-MyHC, whereas V2 and V3 reacted with anti-β-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which
bound anti-α-MyHC antibody while the third bound anti-β-MyHC antibody. We conclude that V1a, V1b, V1c are triplets arising from the dimerization of two distinct α-MyHC isoforms. Hypothyroidism, verified by metabolic studies,
decreased α-MyHC content significantly (t-test, P < 0.001) from 91.6 ± 5.9% (SEM, n = 4) in control animals to 67.2 ± 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in β-MyHC content. We conclude that in adult marsupials, ventricular
myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular
mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians. 相似文献
7.
A protein that copurifies with the bovine prostaglandin F2α receptor (FP) has been isolated and the corresponding rat cDNA has been cloned. Transfection experiments suggest that this
protein inhibits binding of [3H]prostaglandin F2α ([3H]PGF2α) to FP. Histologically, this protein (FP regulatory protein or FPRP) shows a distribution coinciding well with those cells
and tissues that respond to PGF2α. A portion of the 3′ untranslated region of the human homolog to fprp was subcloned, sequenced, and oligonucleotide primers
chosen that allow polymerase chain reaction (PCR) amplification specifically of the human fprp sequence. These primers were
then used in a PCR-based mapping protocol. The human fprp gene was first localized through human/rodent somatic cell hybrids
to human chromosome 1 (100% concordance), and further through yeast artificial chromosome (YAC) pools to region 1p13.1-q21.3
(level 1 mapping). In view of the specific histologic localization of this negative regulator, possible pathological conditions
are mentioned that may cosegregate with this chromosomal region.
Received: 25 May 1995 / Revised: 2 October 1995 相似文献
8.
The connection between three light responses of green leaf cells-membrane potential (Vm), H+ net efflux and growth, was analyzed. Illumination of mesophyll cells in leaves from Argenteum peas caused two rapid responses:
(i) a de- and repolarization of Vm and (ii) an alkalinization of the apoplast. The rapid responses were completely eliminated by the photosynthetic inhibitor
3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) but not affected by ortho-vanadate, an inhibitor of the plasma membrane (PM) H+-ATPase. The rapid changes were followed by a set of delayed responses: (i) a slow, gradual hyperpolarization of Vm, (ii) a gradual acidification of the mesophyll apoplast and (iii) an increased rate of elongation. These three light responses
persisted under DCMU but were completely eliminated by vanadate. The data show that the delayed (in contrast to the rapid)
responses were due to a stimulation of PM H+ pumps which occurred independently of non-cyclic photosynthetic electron transport and the “dark” processes depending on
it. When the rapid responses were blocked by DCMU, light-induced acidification, hyperpolarization of the membrane potential
and growth proceeded simultaneously. A shared (4-min) lag phase indicated slower signal processing in mesophyll than in epidermal
cells where light stimulation of PM H+ pumps was rapid.
Received: 3 September 1998 / Accepted: 15 October 1998 相似文献
9.
Hoh JF Kim Y Lim JH Sieber LG Lucas CA Zhong WW 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2007,177(2):153-163
Cardiac myosins and their subunit compositions were studied in ten species of marsupial mammals. Using native gel electrophoresis,
ventricular myosin in macropodoids showed three isoforms, V
1, V
2 and V
3, and western blots using specific anti-α- and anti-β-cardiac myosin heavy chain (MyHC) antibodies showed their MyHC compositions
to be αα, αβ and ββ, respectively. Atrial myosin showed αα MyHC composition but differed from V
1 in light chain composition. Small marsupials (Sminthopsis crassicaudata, Antechinus stuartii, Antechinus flavipes) showed virtually pure V
1, while the larger (1–3 kg) Pseudocheirus peregrinus and Trichosurus vulpecula showed virtually pure V
3. The five macropodoids (Bettongia penicillata, Macropus eugenii, Wallabia bicolour, M. rufus and M.
giganteus), ranging in body mass from 2 to 66 kg, expressed considerably more α-MyHC (22.8%) than expected for their body size. These
results show that cardiac myosins in marsupial mammals are substantially the same as their eutherian counterparts in subunit
composition and in the correlation of their expression with body size, the latter feature underlies the scaling of resting
heart rate and cardiac cross-bridge kinetics with specific metabolic rate. The data from macropodoids further suggest that
expression of cardiac myosins in mammals may also be influenced by their metabolic scope. 相似文献
10.
G. Schwarz W. Michalek V. Mohler G. Wenzel A. Jahoor 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(3-4):521-530
The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F2 individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly
linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers
and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000
yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was
mapped distal to the Mla locus.
Received: 17 July 1998 / Accepted: 9 August 1998 相似文献
11.
Development of a SCAR (sequence characterised amplified region) marker for molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus L. 总被引:1,自引:0,他引:1
P. Barret R. Delourme N. Foisset M. Renard 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(5-6):828-833
A SCAR (sequence characterised amplified region) has been developed for optimal tagging of the dwarf Bzh gene in Brassica napus. A RAPD marker named OPMO7-730 was previously found closely linked to the dwarf locus at 0.8±0.7 cM. The DNA band corresponding
to this marker was cloned and sequenced, and specific PCR primers were designed. The PCR test allowed us to amplify the locus
corresponding to OPM07-730. With a restriction endonuclease digest and optimal electrophoresis conditions, three allelic forms
of this marker have been recovered on the 40 B. napus accessions tested. The usefullness of this marker in breeding dwarf rapeseed lines is discussed.
Received: 20 April 1998 / Accepted: 29 April 1998 相似文献
12.
R. Bernardo W. E. Nyquist 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(1-2):116-121
Breeders desire populations with a high mean performance and a large genetic variance. Theory and methods are lacking for
predicting additive variance (V
A
) and testcross variance (V
T
) in biparental populations. Breeders have unsuccessfully attempted to predict V
A
based on the coefficient of coancestry ( f ) or molecular-marker similarity between parents. In this paper, we derive the expected values of V
A
and V
T
in biparental populations, examine the variability of V
A
among biparental crosses, and discuss how V
A
and V
T
may be predicted in applied breeding programs. Suppose i is a recombinant inbred derived from the cross between inbreds P
1 and P
2, and inbred j is not a direct descendant of i. Let V
A(i,j)
be the additive variance in the F2 of the (i×j) biparental cross. Let V
T(i, j)
be the variance among testcrosses of F2 individuals with a specific unrelated inbred or population. Assuming linkage equilibrium and the absence of epistasis, V
A(i, j)
=λV
A(P1, j)
+(1−λ) V
A(P2, j)
, where λ= parental contribution of P
1 to i. Similarly, V
T(i, j)
= λV
T(P1, j)
+(1−λ) V
T(P2, j)
. Additive variance in crosses between recombinant inbreds cannot be modelled as a function of f if, as indicated in the literature, V
A
differs among crosses of founder inbreds. If molecular-marker similarity between parents is used as an estimate of f, then a strong linear relationship is likewise not expected between V
A
and marker similarity. Differences between the actual and expected λ led to variation in V
A
. In applied breeding programs, modelling V
A
or V
T
in biparental crosses may be feasible with estimates of V
A
or V
T
in prior crosses and information on λ obtained from molecular-marker data.
Received: 23 September 1997 / Accepted: 30 December 1997 相似文献
13.
Hiroaki Nobuhara Keisuke Kuida Makoto Furutani Toshihiko Shiroishi Kazuo Moriwaki Yusuke Yanagi Tomio Tada 《Immunogenetics》1989,30(6):405-413
Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction
fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains
(C
α,C
β, andC
γ) and a variable region family of the β chain (V
β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC
α,C
β andV
β8 loci and one of three types for theC
γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of
laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have
inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies. 相似文献
14.
David J. Vance Jacqueline M. Tremblay Nicholas J. Mantis Charles B. Shoemaker 《The Journal of biological chemistry》2013,288(51):36538-36547
In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH “heterodimers.” As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics. 相似文献
15.
Kim Y Lucas CA Zhong WW Hoh JF 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2007,177(6):701-705
Ventricular myosin in eutherian mammals undergoes a perinatal change in response to a sharp rise in thyroid hormone levels
during development. In this investigation, changes in ventricular myosin heavy chains (MyHCs) of the tammar wallaby (Macropus eugenii) from early pouch life to adulthood were analysed using native gel electrophoresis, SDS-PAGE and western blotting. Adult
wallaby ventricle showed three myosin isoenzymes, V1, V2 and V3; western blots using specific anti-α-MyHC and anti-β-MyHC antibodies showed their MyHC compositions to be αα, αβ and ββ,
respectively. Ventricular muscle in early pouch joeys expressed predominantly β-MyHC. Up to 200 days, the time of initial
pouch exit, α-MyHC content was around 5%. Thereafter, there was a sharp increase of α-MyHC expression to 35% by 242 days of
age, eventually falling back to 23% in the adult. These changes correlate with known surges in plasma levels of thyroid hormones
around pouch exit. The results suggest that ventricular myosins in a marsupial mammal also undergo a developmental change,
and that marsupial ventricular myosins are thyroid responsive as in eutherians. The increased α-MyHC expression empowers the
heart to meet the enhanced cardiovascular demands of out-of-pouch activity and the thermogenic action of thyroid hormones. 相似文献
16.
The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified CaV1.2 (α1C) and CaV2.3 (α1E) channels. Mibefradil inhibition of peak Ba2+ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response
curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Cav1.2 currents (IC
50) and from IC
50= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for CaV2.3 currents. In the presence of mibefradil, CaV1.2 and CaV2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil
could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation
and drug sensitivity, mibefradil inhibition was studied in inactivation-altered CaV1.2 and CaV2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of
the I–II linker from CaV1.2 in the CaV2.3 host channel), in EC(AID)EEE (part of the I–II linker from CaV1.2 in the CaV2.3 host channel) as well as in CaV1.2 E462R, and CaV2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC
50= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC
50= 41 ± 5 μm (n= 4) and IC
50= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and CaV2.3, whereas intermediate-inactivating channel kinetics (CEEE, CaV1.2 E462R, and CaV1.2 E462K) were inhibited by similar concentrations of mibefradil with IC
50≈ 55–75 μm. The slower CaV1.2 wild-type and CaV1.2 Q473K channels responded to higher doses of mibefradil with IC
50≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (CaV1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (CaV2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca2+ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with
a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel.
Received: 14 March 2001/Revised: 25 June 2001 相似文献
17.
Shi H Yin X Wu M Tang C Zhang H Li J 《Journal of industrial microbiology & biotechnology》2012,39(2):347-357
Using 3′ and 5′ rapid amplification of cDNA ends methods, the full-length cDNA sequence encoding an endo-1,4-β-glucanase of Aspergillus usamii E001 (abbreviated as AuCel12A) was amplified from the total RNA. The clone cDNA sequence of the gene encoding the AuCel12A,
named as Aucel12A, is 1,027 bp in length harboring 5′ and 3′ non-coding regions, as well as a 720 bp of open reading frame that encodes a 16-aa
signal peptide, and a 223-aa mature AuCel12A with a theoretical M.W. of 24,294 Da, a calculated pI of 4.15, and one putative N-glycosylation site. The complete DNA sequence of the gene Aucel12A was amplified from the genomic DNA of A. usamii E001 by using the conventional PCR and pUCm-T vector-mediated PCR initially developed in our lab. The clone DNA sequence
is 1,576 bp in length, consisting of a 5′ flanking regulatory region, three exons, and two introns with sizes of 50 and 66 bp.
The cDNA fragment encoding the mature AuCel12A was expressed in a fully active form in Pichia pastoris. One P. pastoris transformant expressing the highest recombinant AuCel12A (rAuCel12A) activity, labeled as P. pastoris GSCel2-1, was chosen for subsequent studies. Integration of the Aucel12A into P. pastoris genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS-PAGE and enzyme activity assays demonstrated that the rAuCel12A, a glycosylated protein with an apparent M.W.
of 27.0 kDa and a carbohydrate content of 4.82%, was secreted into the culture medium. The purified rAuCel12A displayed the
highest activity at pH 5.0 and 60°C. It was highly stable at a pH range of 3.5–7.0, and at a temperature of 55°C or below.
Its activity was not significantly affected by an array of metal ions and EDTA, but inhibited by Ag+, Hg2+ and Fe2+. The K
m and V
max of the rAuCel12A, towards carboxymethylcellulose-Na (CMC-Na) at pH 5.0 and 50°C were 4.85 mg/ml and 160.5 U/mg, respectively. 相似文献
18.
Purification and characterization of an ADP-dependent phosphofructokinase from Thermococcus zilligii 总被引:1,自引:0,他引:1
R. S. Ronimus Jurre Koning H. W. Morgan 《Extremophiles : life under extreme conditions》1999,3(2):121-129
The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg−1. The enzyme required Mg2+ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH
optimum of 6.4), and the apparent K
m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V
max of 243 U mg−1) and 1.47 mM (apparent V
max of 197 U mg−1), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent
cations including Na+ and K+. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal
reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K
m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V
max of 2.9 U mg−1) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared
to ATP and pyrophosphate-dependent phosphofructokinases.
Received: August 14, 1998 / Accepted: December 2, 1998 相似文献
19.
6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg.
On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed.
The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q10 and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium
Random Bi Bi” kinetic model with Km values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/Vm versus 1/6PG and 1/NADP plots gave a Vm value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a Ki of 31.91±1.31 μM. The relatively small Ki for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD
and 6PGD. 相似文献
20.
A simple method for measuring the settling velocity (V
s) distribution of pollen and spores 30–100 μm in diameter is detailed and evaluated. The method is called the ‘settling tower'
and consists in taking sequential pictures of particles falling under gravity in calm air. The scene is illuminated by a cold
light source, while a camera takes 15 pictures per second. Between 20,000 and 100,000 images are analysed to obtain the distribution
of V
s for a given set of particles. The method was validated using two standard particles with mean diameters of 68 and 108 μm,
respectively, as well as Lycopodium spores, with a mean diameter of 35 μm. For each set of particles, the theoretical V
s distribution was estimated from the particle diameter distribution and the volumetric mass using a non-Stokian law, as the
Reynolds numbers of the particles were large. The mean V
s was measured with the ‘settling tower' with less than 12% error, while the standard deviation of the V
s distribution was estimated with less than 51% error. The maximum error on the mean V
s was 12% for the Lycopodium spores and less than 2% for the two larger particles. The mean V
s of Lycopodium spores was 4.2 cm s−1, and its standard deviation was 0.7 cm s−1. The reason for the small overestimation of V
s for Lycopodium spores by the ‘settling tower' method is discussed. Preliminary measurements shows that, the ‘settling tower' could be of
great practical interest for measuring the distribution of V
s of maize pollen as well as other types of pollen or spores. 相似文献