共查询到20条相似文献,搜索用时 15 毫秒
1.
A. F. Izmailov M. V. Kiselev A. V. Vakurov A. K. Gladilin A. V. Levashov 《Applied Biochemistry and Microbiology》2000,36(1):58-62
Immobilization of α-chymotrypsin on magnetic particles with stable coat with titanium oxides as a main constituent allowed
the biocatalytic system to be quickly and qualitatively separated into the components after completion of the enzymatic reaction.
X-ray phase analysis demonstrated that the coat of magnetic particles is composed mainly of titanium dioxide in brookite modification.
The maximal capacity of the particles amounted to 0.3 mg protein/mg particles. It was demonstrated that the reaction catalyzed
by immobilized α-chymotrypsin proceeds in a kinetic mechanism due to a high dispersion of the ferromagnetic particles. The
catalytic constant (25 s−1) andK
M (0.17 mM) for the immobilized enzyme for the hydrolysis of N-acetyl-L-tyrosine ethyl ester are comparable to the corresponding
characteristics for the free enzyme. 相似文献
2.
Mauro Angeletti Giulio Lupidi Anna Maria Eleuteri Rosalia Tacconi Evandro Fioretti M. Coletta 《Journal of biological inorganic chemistry》1997,2(3):320-326
The effect of aluminum ions on the binding properties of α-chymotrypsin has been studied. The results show that aluminum
does not affect the catalytic rate constant k
cat, but it acts as an enzyme activator favoring the binding of the substrate to the catalytic site (i.e. decreasing K
m). Furthermore, aluminum binding to α-chymotrypsin displays about a threefold decrease in its affinity for the macromolecular
inhibitor bovine pancreatic trypsin inhibitor (BPTI). Altogether, the different effect of aluminum on the binding of synthetic
substrates (e.g. N-α-benzoyl-l-tyrosine ethyl ester, BTEE) and macromolecular inhibitors (e.g. BPTI) to α-chymotrypsin suggests the occurrence of an aluminum-linked
conformational change in the enzyme molecule which brings about a marked structural change at the primary and secondary recognition
sites for substrates and inhibitors. The modulative effect exerted by aluminum on the enzyme hydrolytic activity has been
investigated also as a function of pH. The ion-linked effect appears to be dependent on the pH in a complex fashion, which
suggests that aluminum binding is controlled by the protonation of at least two classes of residues on the enzyme molecule.
Received: 5 December 1996 / Accepted: 11 March 1997 相似文献
3.
Summary. The kinetic aspects of the Perinaphthenone-sensitized photooxidation (singlet molecular oxygen [O2 (1Δg)]-mediated) of α-chymotrypsin (α-Chymo) have been studied at pH 8 and pH 11 as well in reverse micelles (RMs) of sodium 1,
4 bis (2-ethylhexyl) sulfosuccinate (AOT) in n-heptane.
The rate constant values for both overall (kt) and chemical (kr) quenching of O2 (1Δg) by α-Chymo in homogeneous media were higher at pH = 11 than at pH = 8, indicating that the OH-ionized tyrosine (Tyr) residues,
clearly dominate the quenching process. Besides, the rate constants in water were higher than those determined in RMs, demonstrating
that the organized medium protects the protein against photooxidation, probably due to a diminution in both, the accessibility
towards oxidizable amino acid residues and the polarity inside the aggregate, as compared to water. The protection effect
of α-Chymo against the attack by the oxidative species O2 (1Δg) in RMs of AOT seems to be due to the increase of protein stability by the encapsulation within the micellar structure.
The effect of both, surfactant concentration and variation of the ratio ([H2O]/[AOT]) = W on the reactive rate constant was also investigated. The process does not depend significantly on micelles concentration
while the kr values increase as W increases. Furthermore, at W = 30, the highest W studied, kr tends to the value obtained in aqueous medium.
Authors’ address: M. A. Biasutti, Departamento de Química, Campus Universitario, Universidad Nacional de Río Cuarto, (X5804ALH)
Río Cuarto, Argentina 相似文献
4.
Bruylants G Wintjens R Looze Y Redfield C Bartik K 《European biophysics journal : EBJ》2007,37(1):11-18
Protonation/deprotonation equilibria are frequently linked to binding processes involving proteins. The presence of these
thermodynamically linked equilibria affects the observable thermodynamic parameters of the interaction (K
obs, ΔH
obs0). In order to try and elucidate the energetic factors that govern these binding processes, a complete thermodynamic characterisation
of each intrinsic equilibrium linked to the complexation event is needed and should furthermore be correlated to structural
information. We present here a detailed study, using NMR and ITC, of the interaction between α-chymotrypsin and one of its
competitive inhibitors, proflavin. By performing proflavin titrations of the enzyme, at different pH values, we were able
to highlight by NMR the effect of the complexation of the inhibitor on the ionisable residues of the catalytic triad of the
enzyme. Using ITC we determined the intrinsic thermodynamic parameters of the different equilibria linked to the binding process.
The possible driving forces of the interaction between α-chymotrypsin and proflavin are discussed in the light of the experimental
data and on the basis of a model of the complex. This study emphasises the complementarities between ITC and NMR for the study
of binding processes involving protonation/deprotonation equilibria.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
5.
I. Benz W. Beck W. Kraas D. Stoll G. Jung M. Kohlhardt 《European biophysics journal : EBJ》1997,25(3):189-200
Failure of inactivation is the typical response of voltage-gated Na+ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and
antibodies directed against the linker between domains III and IV of the α-subunit. The present patch clamp experiments with cardiac Na+ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na+ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481–1496 of the
cardiac Na+ channel α-subunit) eliminated fast Na+ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process
terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at –40 mV). NBA-modified
and iodate-modified Na+ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain
an only moderate Po of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na+ channels: after cytosolic proteolysis with α-chymotrypsin, trypsin or pronase, mean Po during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently
(time constants 100–200 ms at –40 mV, as a minimum) or was virtually non-operating. In-vitro cleavage of the synthetic linker
sequence 1481–1496 confirmed that this part of the α-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This
iodate resistance indicates that iodate-modified Na+ channels are based on a structural alteration of still another region which is also involved in Na+ inactivation, besides the linker between domains III and IV of the α-subunit. Endogenous peptidases such as calpain did not affect Na+ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na+ channels, mode-switching to a non-inactivating mode.
Received: 25 May 1996 / Accepted: 12 September 1996 相似文献
6.
The complementary fragments of human Hb α, α1–30, and α31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide
splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated
by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction.
The segments α24–30 and α31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous
segment (product) α24–40 has been manipulated by engineering the amino acid replacements to the positions α27 and α31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment α24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies
have confirmed that the side chains at α27 and α31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends
on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute
up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic
stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino
acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the
presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability
to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions
of peptides could be designed into products to facilitate the modular assembly of peptides/proteins. 相似文献
7.
The complementary fragments of human Hb α, α1–30, and α31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide
splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated
by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction.
The segments α24–30 and α31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous
segment (product) α24–40 has been manipulated by engineering the amino acid replacements to the positions α27 and α31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment α24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies
have confirmed that the side chains at α27 and α31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends
on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute
up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic
stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino
acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the
presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability
to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions
of peptides could be designed into products to facilitate the modular assembly of peptides/proteins. 相似文献
8.
H. J. Carlisle T. G. Hales B. A. Schlinger 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1998,182(4):531-538
Songbirds are widely studied to investigate the hormonal control of behavior. However, little is known about the effects
of steroids on neurotransmission in these birds. We used electrophysiological and pharmacological techniques to characterize
γ-aminobutyric acid (GABA) type A receptors (GABAA) of primary cultured telencephalic and hippocampal neurons from developing zebra finches. Additionally, their modulation
by 17β-estradiol(E2), 5α- and 5β-dihydrotestosterone (DHT), 5α- and 5β-pregnan-3α-ol-20-one, and corticosterone was examined. Whole-cell GABA-evoked
currents were inhibited by picrotoxin (10 μmol l−1) and bicuculline methiodide (10 μmol l−1) and potentiated by pentobarbital (100 μmol l−1) and propofol (3 μmol l−1). Loreclezole (10 μmol l−1) potentiated GABA-evoked currents, suggesting the presence of β2, β3 and/or β4 subunits. Diazepam (1 μmol l−1) potentiated currents, while Zn2+ (1 μmol l−1) caused no inhibition, indicating the presence of γ subunits. 5α- and 5β-Pregnan-3α-ol-20-one (100 nmol l−1) potentiated currents, whereas E2 (1 μmol l−1), 5α- and 5β-DHT (1 μmol l−1), and corticosterone (10 μmol l−1) had no detectable effect. We conclude that zebra finch telencephalic and hippocampal GABAA receptors include α, β, and γ subunits and are similar to their mammalian counterparts in both their biophysical and pharmacological
properties. Additionally, GABA-evoked currents are greatly potentiated by 5α- and 5β-pregnan-3α-ol-20-one but show little
or no acute modulation by sex steroids or corticosterone.
Accepted: 12 November 1997 相似文献
9.
A partially purified preparation of the lobster muscle Na+/Ca2+ exchanger was reconstituted with, presumably, random orientation in liposomes. Ca2+ efflux from 45Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular
(ev) medium. Extravesicular Na+ (Na
ev
)-dependent Ca2+ efflux depended directly upon the extravesicular Ca2+ concentration ([Ca2+]
ev
) with a half-maximal activation at [Ca2+]
ev
= 0.6 μm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca2+, as in most other species. In contrast, at low [Na+]
ev
, the Ca
ev
-binding site (i.e., on the cytoplasmic surface) for Ca2+ transported via Ca2+/Ca2+ exchange was half-maximally activated by about 7.5 μm Ca2+. Mild proteolysis of the Na+/Ca2+ exchanger by α-chymotrypsin also upregulated the Na
ev
-dependent Ca2+ efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca2+. Proteolytic digestion in the presence of 1.9 μm free ev Ca2+, however, induced only a 1.6-fold augmentation of Ca2+ efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca2+ also inhibited proteolytic degradation of the Na+/Ca2+ exchanger measured by immunoblotting. These data suggest that Ca2+, bound to a high affinity binding site, protects against the activation of the Na+/Ca2+ exchanger by α-chymotrypsin. Additionally, we observed a 6-fold increase in the Na+/Ca2+ exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster
body fluids.
Received: 20 October 1999/Revised: 13 January 2000 相似文献
10.
The α1 subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α2δ and β subunits. α1E channels directed with the expression of Ba2+ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α1E with α2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α1E with β2a shifted the peak current relationship by −10 mV, and strongly reduced Ba2+ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba2+ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β2a and the neuronal α2bδ subunits increased by ≈10-fold whole-cell Ba2+ currents although coinjection with either β2a or α2bδ alone failed to significantly increase α1E peak currents. Coexpression with β2a and α2bδ yielded Ba2+ currents with inactivation kinetics similar to the β2a induced currents, indicating that the neuronal α2bδ subunit has little effect on α1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit
genes. The slower inactivation was unique to α1E/β2a currents. Coexpression with β1a, β1b, β3, and β4, yielded faster-inactivating Ba2+ currents than currents recorded from the α1E subunit alone. Furthermore, α1E/α2bδ/β1a; α1E/α2bδ/β1b; α1E/α2bδ/β3; α1E/α2bδ/β4 channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The
β subunit-induced changes in the properties of α1E channel were comparable to modulation effects reported for α1C and α1A channels with β3≈β1b > β1a≈β4≫β2a inducing fastest to slowest rate of whole-cell inactivation.
Received: 27 March 1997/Revised: 10 July 1997 相似文献
11.
Evelyne Jouvin-Marche Mariza Goncalves Morgado Nikolaus Trede Patrice N. Marche Dominique Couez Isabelle Hue Carine Gris Marie Malissen Pierre-André Cazenave 《Immunogenetics》1989,30(2):99-104
Genomic DNA from a large panel of inbred strains of mice were hybridized sequentially with 15 Vα, 2 Vδ, 1 Cα, and 1 Cδ probes. Most of the Vα probes detected a high degree of plymorphism and have allowed the definition of five mouse T-cell receptor α (Tcr
α) haplotypes. One of these haplotypes (Tcr
α
e
) appears to arise from a recombination between theTcr
α
b
andTcr
α
a
haplotypes, the latter being the most frequently found in the conventional inbred strains. This recombination event clearly
indicates that the members of at least 11 Vα subfamilies are not closely linked but highly interspersed with one another on chromosome 14. 相似文献
12.
Frochot Céline Vanderesse Régis Driou Alain Linden Guy Marraud Michel Thong Cung Manh 《International journal of peptide research and therapeutics》1997,4(4-6):219-225
Summary Using the Boc-strategy, a step-by-step synthesis on the PAM solid support of three aza-, iminoaza- and reduced aza-peptide
homologues is described. From the same hydrazinocarbonyl peptide-PAM precursor, the coupling of either a Boc-amino acid or
a Boc-amino aldehyde gives rise to an aza-peptide or an iminoaza-peptide, containing the Cα-CO-NH-Nα-CO-NH-Cα or Cα-CH=N-Nα-CO-NH-Cα surrogate, of the peptide motif, respetively. In situ reduction of the latter by NaBH3CN leads to a reduced aza-peptide containing the Cα-CH2-NH-Nα-CO-NH-Cα moiety. The key step synthesis of the hydrazinocarbonyl peptide-PAM precursor is carried out by coupling on the growing peptide
chain theN-Boc-azaamino acid chloride obtained by the action of triphosgene on the, correspondingN-Boc-hydrazine. These modifications have been introduced in position 1–2 of the YLGYLEQLLR benzodiazepine-like decapeptide. 相似文献
13.
Unlike the other haemoglobinopathies, few researches have been published concerning α-thalassaemia in Tunisia. The aim of
the present work is to acquire further data concerning α-thalassaemia prevalence and molecular defects spectrum in Tunisia,
by collecting and studying several kinds of samples carrying α-thalassaemia. The first survey conducted on 529 cord blood
samples using cellulose acetate electrophoresis, have displayed the prevalence of 7.38% Hb Bart’s carriers at birth. Molecular
analyses were conducted by PCR and DNA sequencing on 20 families’ cases from the above survey carrying the Hb Bart’s at birth
and on 10 Hb H diseased patients. The results showed six α-globin gene molecular defects and were responsible for α-thalassaemia:
-α3.7, - -MedI, αTSaudi, α2cd23GAG→Stop, Hb Greone Hart: α1119CCT→TCT corresponding to 11 genotypes out of which two are responsible for Hb H disease (- -Med/-α3.7) and (αTSaudiα/αTSaudiα) and a newly described polymorphism: α+6C→G. The geographical repartition of α-thal carriers showed that the -α3.7 deletion is distributed all over the country, respectively the αHphI and αTSaudi seem to be more frequent in the central region of the northeast region. The haematological and clinical data showed a moderate
phenotype with a late age of diagnosis for Hb H disease. This work had permitted, in addition to an overview on α-thalassaemia
in the country, the optimization of protocols for α-thalassaemia detection in our lab, allowing further investigations concerning
phenotype-genotype correlation in sickle cell disease or β-thalassaemia. 相似文献
14.
Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium 总被引:3,自引:1,他引:2
Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies
of the Na/K ATPase. RNase protection assays showed that all cells express α1- and α2-isoforms of the Na/K pump but not the α3-isoform, however the α2-isoform dominates in anterior cells whereas the α1-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the
Na/K pump current (I
P
), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I
P
blockade data indicate the α1-isoform has a dissociation constant of 100 μm DHO whereas for the α2-isoform it is 0.75 μm DHO. Both α1- and α2-isoforms are half maximally activated at an intracellular Na+-concentration of 9 mm. The α1-isoform is half maximally activated at an extracellular K+-concentration of 3.9 mm whereas for the α2-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α1-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α2-isoform. Under normal physiological conditions, I
P
in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer
to the area of cell membrane assuming a capacitance of around 1 μF/cm2. Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm2 at the equator vs. 0.5 μA/cm2 at the anterior pole.
Received: 17 May 2000/Revised: 11 August 2000 相似文献
15.
Olga A. Akimova Johanne Tremblay James W. Van Huysse Pavel Hamet Sergei N. Orlov 《Apoptosis : an international journal on programmed cell death》2010,15(1):55-62
Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood.
This study examines the role of Na+,K+-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing
a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na+,K+-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of 86Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na+,K+-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na+]i/[K+]i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of
mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of
the [Na+]i/[K+]i ratio evoked by Na+,K+-ATPase inhibition in K+-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R
subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na+,K+-ATPase-mediated Na+ and K+ fluxes and inversion of the [Na+]i/[K+]i ratio. 相似文献
16.
Tomoya Fujita James C. Ogbonna Hideo Tanaka Hideki Aoyagi 《Journal of applied phycology》2009,21(2):185-191
The effects of reactive oxygen species (ROS) on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis were investigated. Addition of an organic carbon source to the medium resulted in increased mitochondrial activity, intracellular
O2
- concentration and α-tocopherol productivity in E. gracilis W14ZUL (a chloroplast deficient mutant). α-Tocopherol productivity of the wild-type strain (with both mitochondria and chloroplast)
was higher than that of the W14ZUL strain. In the case of the wild strain, the O2
− generated in chloroplasts was efficiently scavenged by the α-tocopherol synthesized inside the chloroplast. In photoheterotrophic
culture (with an organic carbon source), there was a positive correlation between α-tocopherol production and O2
− generation. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) resulted in increased O2
− generation and α-tocopherol productivity. These results indicate that the ROS generated in mitochondria and chloroplasts
play important roles in α-tocopherol production by E. gracilis. The presence of chloroplasts and generation of intracellular ROS are important for efficient production of α-tocopherol. 相似文献
17.
In this study, human α-1,4-N-acetylglucosaminyltransferase (α4GnT) fused with GFPuv (GFPuv-α4GnT) was expressed using both a transformed cell system and silkworm larvae. A Tn-pXgp-GFPuv-α4GnT cell line, isolated after expression vector transfection, produced 106 mU/ml of α4GnT activity in suspension culture.
When Bombyx mori nucleopolyhedrovirus containing a GFPuv-α4GnT fusion gene (BmNPV-CP
−/GFPuv-α4GnT) bacmid was injected into silkworm larvae, α4GnT activity in larval hemolymph was 352 mU/ml, which was 3.3-fold higher
than that of the Tn-pXgp-GFPuv-α4GnT cell line. With human calnexin (CNX) or human immunoglobulin heavy chain-binding protein (BiP, GRP78) coexpressed under
the control of the ie-2 promoter, α4GnT activity in larval hemolymph increased by 1.4–2.0-fold. Moreover, when BmNPV-CP
−/GFPuv-α4GnT bacmid injection was delayed for 3 h after BmNPV-CP
−/CNX injection, the α4GnT activity increased significantly to 922 mU/ml, which was 8.7-fold higher than that of the Tn-pXgp-GFPuv-α4GnT cell line. Molecular chaperone assisted-expression in silkworm larvae using the BmNPV bacmid is a promising tool for
recombinant protein production. This system could lead to large-scale production of more complex recombinant proteins. 相似文献
18.
The contribution of the allelicMtz
3 andMtz
4 genes to the formation of individual rabbit serum α2-macroglobulin (α2M) molecules was examined by precipitation of α2M from rabbits of known genotype with antiallotype antisera. The α2M was isolated fromz
3z3 andz
4z4 homozygous andz
3z4 heterozygous rabbits, iodinated with I125 and precipitated by sequential reactions with antiallotype antiserum and goat anti-rabbit IgG. Purified unlabeled α2M or α2M in serum was used to inhibit competitively the reaction of antiallotype antiserum and labeled α2M. Nearly all α2M molecules have z3 or z4 antigenic determinants; approximately 50% of α2M molecules in heterozygotes have both. Altogether, the z3, z3,4, and z4 molecules in heterozygotes have approximately 60%
of the number of z3 and 40% of the number of z4 determinants as compared to the respective homozygotes. Unlike all other known
allelic blood protein systems of rabbits, allelic exclusion does not occur in α2M molecules of heterozygotes; rather, hybrid molecules are formed.
Presented in part at the Fifty-fourth and Fifty-fifth Annual Meetings of the Federation of American Societies for Experimental
Biology, Atlantic City, New Jersey, April 12–17, 1970, and Chicago, Illinois, April 12–17, 1971. This investigation was supported
in part by U.S. Public Health Service Grants AI-09241 and AI-07043. B.H.B. performed this investigation in partial fulfillment
of the requirements for the Doctor of Philosophy Degree in the Graduate College; he is supported by a postdoctoral fellowship
from the Schweppe Foundation. K.L.K. is the recipient of U.S. Public Health Service Research Career Development Award AI-28687. 相似文献
19.
The concentration-dependent self-association of α-chymotrypsin is known to be influenced by various factors including the
presence of small molecules and autolysis products. In this connection the effect of various amino acids on the self-association
of α-chymotrypsin has been studied, as a point of interest, by measuring the sedimentation coefficient of α-chymotrypsin.
The influence of an amino acid is seen to be governed by the nature of its side chain. Some amino acids do not affect the
self-association of α-chymotrypsin at all while some affect it moderately and some others considerably. Functional groups
such as the - OH group of Ser or the phenolic ring of Tyr do not seem to influence self-association behaviour. Based on these
effects, amino acids could be categorized into 3 groups. Activity studies in the presence of amino acids indicate that the
site of self-association and the active-site are probably mutually exclusive. 相似文献
20.
Functional activation of α2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [35S]-guanosine 5′-O-(γ-thiotriphosphate) ([35S]GTPγS) binding assay. α2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [35S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However,
even after careful optimization of experimental conditions the specificity of ligands for rat α2 adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems
before achieving their maximal effect in the α2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948)
to inhibit agonist’s effect, allowed us to filtrate out α2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies
of this system for different animals from behavioral experiments. 相似文献