共查询到20条相似文献,搜索用时 15 毫秒
1.
《Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology》1996,1292(1):69-76
The susceptibility of a-lactalbumin to transglutaminase reactions was studied using an enzyme from Streptoverticillium which can catalyze the reactions irrespective of the presence or absence of Ca2+. Transglutaminase-catalyzed polymerization of a-lactalbumin in the native state occurred to a very limited extent. Transformation from the native state to the molten globule state brought about by Ca2+-removal from holo-a-lactalbumin enhanced the polymerization of the protein catalyzed by transglutaminase. The incorporation of Carbobenzoxy-Gln-Gly into a-lactalbumin through the enzyme reaction was investigated to determine the amounts of lysine residues which are present at molecular surface and available to the enzyme. There was no significant difference in the amount of available lysine residues between the native: and the molten globule molecule. However, the amount of surface glutamine residues incorporated with monodansylcadaverine by transglutaminase was remarkably higher in the molten globule state than that in the native state. The monodansylcadaverine-incorporated site of a-lactalbumin in the molten globule state was identified as Gln-54 by amino-acid sequence analysis of fluorescence-labeled peptides separated from chymotryptic digests of the protein. Possible reason for selective labeling of Gln-54 in molten globule a-lactalbumin was proposed. 相似文献
2.
Devaki A. Kelkar Arunima Chaudhuri Sourav Haldar Amitabha Chattopadhyay 《European biophysics journal : EBJ》2010,39(10):1453-1463
The relevance of partially ordered states of proteins (such as the molten globule state) in cellular processes is beginning
to be understood. Bovine α-lactalbumin (BLA) assumes the molten globule state at acidic pH. We monitored the organization
and dynamics of the functionally important tryptophan residues of BLA in native and molten globule states utilizing the wavelength-selective
fluorescence approach and fluorescence quenching. Quenching of BLA tryptophan fluorescence using quenchers of varying polarity
(acrylamide and trichloroethanol) reveals varying degrees of accessibility of tryptophan residues, characteristic of native
and molten globule states. We observed red edge excitation shift (REES) of 6 nm for the tryptophans in native BLA. Interestingly,
we show here that BLA tryptophans exhibit REES (3 nm) in the molten globule state. These results constitute one of the early
reports of REES in the molten globule state of proteins. Taken together, our results indicate that tryptophan residues in
BLA in native as well as molten globule states experience motionally restricted environment and that the regions surrounding
at least some of the BLA tryptophans offer considerable restriction to the reorientational motion of the water dipoles around
the excited-state tryptophans. These results are supported by wavelength-dependent changes in fluorescence anisotropy and
lifetime for BLA tryptophans. These results could provide vital insight into the role of tryptophans in the function of BLA
in its molten globule state in particular, and other partially ordered proteins in general. 相似文献
3.
Wolfgang Pfeil 《Proteins》1998,30(1):43-48
Thermal and denaturant-induced transitions of the acid molten globule state of bovine α-lactalbumin (acid [A] state) are analyzed by scanning calorimetry, titration calorimetry, viscosimetry, and derivative spectroscopy. A denaturant-induced heat effect of the A state is shown by a calorimetric difference titration of the A-state versus unfolded (reduced) α-lactalbumin. However, changes of viscosity and derivative spectra do not parallel the heat effect. At thermal denaturation monitored by derivative spectroscopy and scanning microcalorimetry the presence of a gradual transition in α-lactalbumin A state is shown. The results are consistent with the existence of tertiary interactions in the A state and the absence of a cooperative unfolding transition of the molten globule. The results do not support the idea that the molten globule is a third thermodynamic state. Proteins 30:43–48, 1998. © 1998 Wiley-Liss, Inc. 相似文献
4.
Arunima Chaudhuri 《Biochemical and biophysical research communications》2010,394(4):1082-963
Bovine α-lactalbumin (BLA) is known to be present in molten globule form in its apo-state (i.e., Ca2+ depleted state). We explored the organization and dynamics of the functionally important tryptophan residues of BLA in native, molten globule and denatured states utilizing the wavelength-selective fluorescence approach. We observed red edge excitation shift (REES) of 7 nm for the tryptophans in native BLA. Interestingly, we show here that BLA tryptophans exhibit considerable REES (8 nm) in its molten globule state. Taken together, these results indicate that tryptophan residues in BLA in native as well as molten globule states experience motionally restricted environment. We further show that even the denatured form of BLA exhibits a modest REES of 3 nm, indicating that the tryptophans are shielded from bulk solvent, even when denatured, due to the presence of residual structure around tryptophan(s). This is further supported by wavelength-dependent changes in fluorescence anisotropy and lifetime for BLA tryptophans. These novel results constitute one of the first reports of REES in the molten globule state of proteins, and could provide vital insight into the role of tryptophans in the function of BLA in its molten globule state in particular, and other partially ordered proteins in general. 相似文献
5.
6.
R.E. Fenna 《Journal of molecular biology》1982,161(1):211-215
Crystals of human milk α-lactalbumin have been grown from ammonium sulfate solutions in the presence of calcium ions at 35 °C. They belong to space group P21212, with unit cell dimensions: and have a single molecule in the asymmetric unit. The crystals diffract X-rays strongly to Bragg spacings of 2 Å and appear suitable for X-ray structural analysis. 相似文献
7.
R.E. Fenna 《Journal of molecular biology》1982,161(1):203-210
Three new crystal forms of cow α-lactalbumin are described. A trigonal form in space group P3121 or P3221 has unit cell dimensions: . A hexagonal form in space group P622 has unit cell dimensions: . A second trigonal form grown in the presence of calcium ions belongs to space group P321 with unit cell dimensions: . The significance of these new crystal forms to the structure determination of cow α-laetalbumin is discussed. 相似文献
8.
9.
Summary Whether both casein and noncasein (serum or whey) proteins of milk are contained within the same secretory vesicles of milk secreting mammary epithelial cells was explored. Antibodies to a major casein and to -lactalbumin of rat milk were localized in thin sections with colloidal gold-conjugated second antibodies. Antibodies to the casein component bound to an antigen present within lumina of Golgi apparatus cisternae and within secretory vesicles. This antigen was also recognized in structures within secretory vesicles and within alveolar lumina which were ultrastructurally identified as casein micelles. Antigens recognized by antibodies to -lactalbumin also were present in Golgi apparatus cisternae and within secretory vesicles. Both anti-casein and anti--lactalbumin antibodies recognized antigens within the same secretory vesicles. These observations show that one major noncasein protein of rat's milk is present in casein-containing secretory vesicles. 相似文献
10.
M. P. Thompson H. M. Farrell Jr. Sanjeeva Mohanam Sue Liu W. R. Kidwell M. P. Bansal R. G. Cook D. Medina Claire E. Kotts Mozeena Bano 《Protoplasma》1992,167(3-4):134-144
Summary A growth inhibitory protein, mammary inhibitory activity (MIA), was purified to apparent homogeneity from human milk. At concentrations of 5 to 10 ng/ml, the factor inhibited the growth of mammary epithelial cells by 30–80% and also inhibited the growth of normal rat kidney cells. Whereas the cell division of normal human mammary epithelium in primary culture was inhibited by MIA, cell division by fibroblasts from the same tissues was unresponsive. Inhibition was dose and time dependent and readily reversed when MIA was removed. MIA also inhibited growth in culture for three cell lines. The growth inhibitory protein migrated as a 14 kDa protein under reducing conditions on polyacrylamide gels in the presence of sodium dodecyl sulfate. The apparent isoelectric point was pI 5.0. The amino acid composition of MIA resembled that of -lactalbumin, and sequence analysis of the N-terminal region comprising residues 1–24 and an isolated peptide were identical with the N-terminal and residues 66–81 of human -lactalbumin. In addition, MIA was active in the lactose synthase system. The results strongly suggest that MIA and -lactalbumin are identical proteins. Consistent with these results, -lactalbumin preparations from several mammalian species, including human, goat, cow and camel, were all found to be growth inhibitory for cultured mammary epithelial cells. The inhibitory activity associated with human -lactalbumin was destroyed by digestion with pepsin or chymotrypsin, by carboxymethylation of cysteine, or by cleavage of methionine 90 following cyanogen bromide treatment. The results raise the possibility that during lactation -lactalbumin, a product of mammary cell differentiation, could be a physiologically relevant feed-back inhibitor of mammary cell growth and perhaps of other cell types as well.Abbreviations MIA
mammary inhibitory activity
- MDGI
mammary derived growth inhibitor
- -LA
alpha lactalbumin
- H--LA
human -lactalbumin
- NRK
normal rat kidney
- IMEM
improved minimal essential medium
- DMEM
Dulbecco's modified Eagles medium
- FCS
fetal calf serum
- EGF
epidermal growth factor
- TGF
transforming growth factor
- CNBr
cyanogen bromide
- SDS
sodium dodecyl sulfate
- kDa
kilodaltons
- ND-PAGE
non-denaturing polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid 相似文献
11.
R. N. Ma C. J. Deng X. M. Zhang X. P. Yue X. Y. Lan H. Chen C. Z. Lei 《Molecular Biology》2010,44(4):536-540
The α-lactalbumin (α-LA) plays a key role in lactose synthesis in mammary glands of domestic animals. Mutations in the α-LA gene are associated with the milk traits in dairy cattle. In our study, a novel SNP: NO_X06366: g.875 C > T was detected in 708 dairy goat individuals—268 of the Xinong Saanen breed and 440 of Guanzhong breed, which revealed a synonymous
mutation in the exon 1 of α-LA gene. The Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) and sequencing techniques showed that
there were three genotypes: CC, CT and TT. Moreover, the χ2-test showed that the genotype frequencies of the two breeds were in good agreement with the Hardy-Weinberg equilibrium (P > 0.05). The relationship of the polymorphism of dairy goat α-LA gene with the milk trait and the body size trait was revealed. Individuals with the CC genotype were significantly smaller at chest circumference than those with CT (P < 0.05) in both breeds. But the milk trait and other body size traits of the two dairy goat breeds had no significant association
with genotypes studied. 相似文献
12.
Md. Khurshid Alam Khan Md. Hamidur Rahaman Md. Imtaiyaz Hassan Tej P. Singh Ali A. Moosavi-Movahedi Faizan Ahmad 《Journal of biological inorganic chemistry》2010,15(8):1319-1329
It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of
this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue
ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured
by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan
fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude
that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability
of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more
stable than the PMG state by 5.4 ± 0.1 kcal mol−1, whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol−1. A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the
PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential
scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states. 相似文献
13.
LIU Siguo WEI Yingyun HU Guofa GAO Hong LIU Sijin LAO Weide 《中国科学:生命科学英文版》2004,47(3):197-202
Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a 2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of the hα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost more milk output. 相似文献
14.
The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II α-mannosidase from Canavalia ensiformis (Jack Bean) (Jbα-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jbα-man in the vicinity of 1 M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jbα-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60 °C and stability of the structure up to 80 °C unlike the native Jack Bean α-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II α-mannosidase. 相似文献
15.
Five female transgenic mice were produced by microinjection using a construct made up of a 7.3-kb-5′ flanking region and a
2.0-kb coding region of human α-lactalbumin, as well as a 227-bp 3′-flanking region from bovine growth hormone gene. A founder
female expressed human α-lactalbumin as much as 0.3 g per liter of its milk, approximately a 3-fold increase in the total
α-lactalbumin concentration of the transgenic mouse milk. Compared with the normal mice, the expression profile of thehα-Lac transgene in the transgenics is different during the lactation, showing low level in the first 3 days and becoming increased
from day 4, then gradually reaching and stabilizing at the highest level from day 13. In addition, the milk yielding volume
in the transgenics tended to be higher than in normal mice, suggesting higher concentrations of α-lactalbumin might boost
more milk output. 相似文献
16.
LIU Siguo WEI Yingyun HU Guofa GAO Hong LIU Sijin & LAO Weide Institute of Genetics Developmental Biology Chinese Academy of Sciences Beijing China 《中国科学:生命科学英文版》2004,(3)
a-lactalbumin(a-Lac),amajorwheyprotein,isacalciummetalloprotein,thathasbeenfoundinallmilksstudiedsofar.ItinteractswithUDP-galactosyl-transferasetoformthelactosesynthetaseandthusmightbeakeyproteinforlactogenesis.Lactosesyn-thetaseispostulatedtobetherate-limitingenzymeforlactosebiosynthesis.Theincreaseda-Lacactivitycanproducesufficientlactosesynthetaseforthesynthesisoflactose,andinmilkyieldbydrawingwaterintomilk,sincelactoseisanosmoreactivemolecule.Transgenicswineoverexpressingbovinea-lactalbu… 相似文献
17.
Cristiana Faria Carla D. Jorge Nuno Borges Sandra Tenreiro Tiago F. Outeiro Helena Santos 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Protein aggregation in the brain is a central hallmark in many neurodegenerative diseases. In Parkinson's disease, α-synuclein (α-Syn) is the major component of the intraneuronal inclusions found in the brains of patients. Current therapeutics is merely symptomatic, and there is a pressing need for developing novel therapies. Previously we showed that mannosylglycerate (MG), a compatible solute typical of marine microorganisms thriving in hot environments, is highly effective in protecting a variety of model proteins against thermal denaturation and aggregation in vitro.Methods
Saccharomyces cerevisiae cells expressing eGFP-tagged α-Syn, were further engineered to synthesize MG. The number of cells with fluorescent foci was assessed by fluorescence microscopy. Fluorescence spectroscopy and transmission electron microscopy were used to monitor fibril formation in vitro.Results
We observed a 3.3-fold reduction in the number of cells with α-Syn foci and mild attenuation of α-Syn-induced toxicity. Accordingly, sucrose gradient analysis confirmed a clear reduction in the size-range of α-Syn species in the cells. MG did not affect the expression levels of α-Syn or its degradation rate. Moreover, MG did not induce molecular chaperones (Hsp104, Hsp70 and Hsp40), suggesting the implication of other mechanisms for α-Syn stabilization. MG also inhibited α-Syn fibrillation in vitro.Conclusions
MG acts as a chemical chaperone and the stabilization mechanism involves direct solute/protein interactions.General significance
This is the first demonstration of the anti-aggregating ability of MG in the intracellular milieu. The work shows that MG is a good candidate to inspire the development of new drugs for protein-misfolding diseases. 相似文献18.
Zhao M Cascio D Sawaya MR Eisenberg D 《Protein science : a publication of the Protein Society》2011,20(6):996-1004
Aggregates of the protein α-synuclein are the main component of Lewy bodies, the hallmark of Parkinson's disease. α-Synuclein aggregates are also found in many human neurodegenerative diseases known as synucleinopathies. In vivo, α-synuclein associates with membranes and adopts α-helical conformations. The details of how α-synuclein converts from the functional native state to amyloid aggregates remain unknown. In this study, we use maltose-binding protein (MBP) as a carrier to crystallize segments of α-synuclein. From crystal structures of fusions between MBP and four segments of α-synuclein, we have been able to trace a virtual model of the first 72 residues of α-synuclein. Instead of a mostly α-helical conformation observed in the lipid environment, our crystal structures show α-helices only at residues 1-13 and 20-34. The remaining segments are extended loops or coils. All of the predicted fiber-forming segments based on the 3D profile method are in extended conformations. We further show that the MBP fusion proteins with fiber-forming segments from α-synuclein can also form fiber-like nano-crystals or amyloid-like fibrils. Our structures suggest intermediate states during amyloid formation of α-synuclein. 相似文献
19.
Yukihiko Hirai Eugene A. Permyakov Lawrence J. Berliner 《Journal of Protein Chemistry》1992,11(1):51-57
The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR. 相似文献
20.
Victoria A. Higman Heike I. Rösner Raffaella Ugolini Lesley H. Greene Christina Redfield Lorna J. Smith 《Journal of biomolecular NMR》2009,45(1-2):121-131
Backbone 15N relaxation parameters and 15N–1HN residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. 15N R2 values in some regions of all-Ala α-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 310 helices in native α-LA show R2 values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101–106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala α-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant. 相似文献