Biomedical subjects

E A Permyakov

Publications and source records attributed to E A Permyakov.

At least 37 records · Page 2Linked to original sources

PubMed1992

Proteolytic digestion of alpha-lactalbumin: physiological implications.

The kinetics of the partial digestion of bovine alpha-lactalbumin (alpha-LA) by trypsin, alpha-chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of alpha-LA to trypsin and chymotrypsin at 37 and 5 degrees C decrease in the following order: Ca(II)-alpha-LA greater than Zn(II), Ca(II)-alpha-LA greater than apo-alpha-LA. The HPLC digestion patterns of Ca(II)-alpha-LA and Zn(II), Ca(II)-alpha-LA at 5 and 37 degrees C were similar, while the corresponding digestion patterns for apo-alpha-LA were quite different, reflecting the existence of the thermally induced denaturation states of apo-alpha-LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded alpha-LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37 degrees C due to Zn(II)-induced shift of the thermal transition of alpha-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 alpha-LA. The acidic form of alpha-LA (at pH 2.2 and 37 degrees C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or alpha-chymotrypsin at neutral pH. Complexation of alpha-LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to alpha-LA may have physiological significance (e.g., for nutritional transport).

Animals↗

PubMed1992

Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.

Animals↗

PubMed1992

Multi-stage nature of the thermal denaturation process in collagen.

Thermal denaturation of acid-soluble collagen from polar cod (Eleginus gracialis) skin has been studied by scanning microcalorimetry and intrinsic spectrofluorimetry methods. The thermal denaturation process occurs in three independent stages reflecting the melting of 33 kDa, 97 kDa, and 230 kDa domains. Thermodynamical parameters of the collagen denaturation have been determined.

Animals↗

PubMed1991

Noncovalent complex between domain AB and domains CD*EF of parvalbumin.

The interaction between domain AB and domains CD*EF of pike parvalbumin III has been studied by intrinsic fluorescence spectroscopy. In the presence of Ca2+ ions, parvalbumin fragment 38-108 containing two calcium binding sites interacts with the short peptide 1-37 with association constant 10(5.3 +/- 0.5) M-1. Removal of Ca2+ ions results in the disappearance of the interaction. The affinity of the complex of the two fragments for calcium is 50-times higher than the affinity of the isolated fragment 38-108, but slightly lower than that of the intact protein.

Binding Sites↗

PubMed1991

Binding of Zn(II) ions to alpha-lactalbumin.

The binding of Zn(II) ions to human and bovine alpha-lactalbumin has been studied by fluorescence, scanning microcalorimetry, and proteolytic digestion. The intrinsic tryptophan fluorescence spectrum of Ca(II)-loaded alpha-lactalbumin is insensitive to Zn(II) binding to the strong cation binding sites (Zn:protein ratios up to 20), yet the thermal denaturation transition, as detected by intrinsic fluorescence, is shifted toward lower temperatures. On the other hand, low concentrations of Zn(II) ([Zn]:[protein] less than 1) shift heat sorption curves toward lower temperatures. It was concluded that alpha-lactalbumin possess several relatively strong Zn(II) binding sites, which are filled sequentially, the process being accompanied by protein aggregation. The strongest Zn(II) binding (5 x 10(5) M-1) increases its susceptibility to tryptic and chymotryptic digestion, slightly decreases its affinity for the fluorescent probe, bis-ANS, and alters its interactions with UDP-galactose. Zn(II) binding to aggregated forms of alpha-lactalbumin increases its affinity to bis-ANS.

Animals↗

PubMed1991

Calcium-regulated interactions of human alpha-lactalbumin with bee venom melittin.

Affinity chromatography, fluorescence and circular dichroism spectroscopy methods have been used to study the interaction of melittin, a 26-residue peptide from bee venom, with Ca2(+)-binding alpha-lactalbumin from human milk. It has been revealed that melittin binds to the apo- and acidic states of alpha-lactalbumin while the presence of Ca2+ makes the interaction essentially weaker. The association constant for the complex of melittin with apo-alpha-lactalbumin determined from spectropolarimetric melittin-titration data is 2 X 10(7) M-1. The complexation of alpha-lactalbumin with melittin decreases its affinity to Ca2+ by three orders of magnitude. The interaction of apo-alpha-lactalbumin with melittin causes some changes in the environment of its aromatic amino acid residues and drastically alters the conformation of melittin, increasing its alpha-helical content but leaving its single tryptophan residue accessible to water. In the case of the acidic state of alpha-lactalbumin the interaction does not induce an increase in alpha-helical content of melittin.

Binding Sites↗

PubMed1991

Spectrofluorimetric studies on C-terminal 34 kDa fragment of caldesmon.

Analysis of the tryptophan fluorescence emission spectra of caldesmon and its 34 kDa C-terminal fragment indicates that all tryptophan residues are located on the surface of the molecule, accessible to solvent. All three tryptophan residues of the 34 kDa fragment and four of the five tryptophan residues of intact protein are accessible to free water, whereas one located in the N-terminal region of molecule is accessible only to bound water molecules. The temperature dependence of the fluorescence parameters indicates higher thermal stability of the 34 kDa fragment than the whole caldesmon molecule. The interaction of the 34 kDa fragment of caldesmon (like that of the intact molecule) with calmodulin is accompanied by a blue shift of the fluorescence emission maximum and an increase in the relative quantum yield. Computer-calculated binding constants show that the binding of calmodulin to the 34 kDa fragment (K = 2.5 x 10(5) M-1) is of two orders of magnitude weaker than that to intact caldesmon (K = 1.4 x 10(7) M-1). The interaction with tropomyosin results in a blue shift of the spectrum of the 34 kDa fragment, yet there is no effect on the spectrum of intact caldesmon. Binding constants of tropomyosin to caldesmon (K = 3.8 x 10(5) M-1) and its 34 kDa fragment (K = 2.3 x 10(5) M-1) are similar. Binding of calmodulin to caldesmon and to the 34 kDa fragment affects their interaction with tropomyosin.

Animals↗

PubMed1991

The effect of alkali light chains on the thermal stability of myosin subfragment 1.

Thermal denaturation of myosin subfragment 1 (S1) isoforms from rabbit skeletal muscle containing the different alkali light chains A1 and A2 [S1(A1) and S1(A2), respectively] were studied by various methods. Turbidity measurements showed that thermally induced (heating rate 1 degrees C min-1) aggregation of S1(A1) occurs at lower temperatures than that of S1(A2). However, the temperature dependences of the tryptophan fluorescence spectrum and that for ATPase inactivation were the same for S1(A1) and S1(A2). Thermal denaturation of the S1 isoforms was also studied by differential scanning microcalorimetry with the 'successive annealing' method. Three independently melting cooperative regions (domains) were revealed in the molecules of both isoforms. Heat sorption curves for the S1 isoforms were different only for the most thermolabile domain, which had a maximum at 36 degrees C for S1(A1) and at 40.5 degrees C for S1(A2). Two other peaks had maxima at 46-47 degrees C and 50-51 degrees C for both isoforms. It is proposed that alkali light chains A1 and A2 differently affect the conformation of the most thermolabile domain, which probably does not contain trytophan residues and does not take part directly in the formation of the active site of the S1 ATPase.

Animals↗

PubMed1990

Domain structure of myosin subfragment-1. Selective denaturation of the 50 kDa segment.

The structure of the myosin subfragment-1 (S1) from rabbit skeletal muscle was studied using differential scanning microcalorimetry. Three independently melting regions (domains) were revealed in S1. Selective denaturation of the middle 50 kDa segment of the S1 heavy chain resulted in the disappearance of the heat sorption peak corresponding to the melting of the first, the most thermolabile domain without any effect on the thermally induced blue shift of the intrinsic tryptophan fluorescence spectrum which occurs within the temperature region of melting of the second domain. It is concluded that the most thermolabile domain seems to correspond to the N-terminal part of the 50 kDa segment devoid of tryptophan residues.

Animals↗

PubMed1990

Some aspects of fluorescence and microcalorimetric studies of cytochrome C oxidase.

Effects of the solubilizing detergent type, pH and temperature on the structure of cytochrome c oxidase have been studied by the intrinsic fluorescence and scanning microcalorimetry methods. The data obtained allow to conclude that the enzyme solubilization by lauryl maltoside gives a more native preparation in comparison with that obtained by solubilization in Tween 80.

Animals↗

PubMed1988

Study of pH- and temperature-induced transitions in F-protein (phosphofructokinase) by spectroscopy and microcalorimetry methods.

The temperature- and pH-induced transitions in F-protein (phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11] have been studied by means of microcalorimetry and fluorescence and CD spectroscopy. An increase in pH from approx. 6.0 to approx. 8.0 causes a change in the protein state which seems to correspond to a shift of the dimer-tetramer equilibrium in favour of the tetramers. In the absence of phosphate, stability of the protein to temperature- and urea-induced denaturation at pH 6.0 is higher than that at pH 8.0. An addition of 150 mM phosphate results in a pronounced increase in the protein's stability in such a way that the protein becomes more stable at pH 8.0 than at pH 6.0. The shift of the denaturational heat capacity peak induced by the phosphate binding exceeds 25 degrees C at pH 8.0 and 9 degrees C at pH 6.0.

Animals↗

PubMed1988

Environment of tryptophan residues in various conformational states of alpha-lactalbumin studied by time-resolved and steady-state fluorescence spectroscopy.

Decay curves for tryptophan fluorescence of bovine and human alpha-lactalbumin in different states (metal-free and Ca2+ or Mg2+-loaded states of the native and thermally denatured proteins) have been measured at different wavelengths. The curves are best fitted by a sum of three exponents assigned to emission of individual tryptophan residues. The results suggests that the red shift of the fluorescence spectrum of alpha-lactalbumin caused by release of the bound Ca2+ or thermal denaturation is due to changes in the environment of all emitting tryptophan residues.

Animals↗

PubMed1988

Interaction of alpha-lactalbumin with Cu2+.

It has been shown by intrinsic fluorescence spectroscopy that alpha-lactalbumin has several Cu2+ -binding sites per molecule. The Ca2+ -loaded protein binds two or more Cu2+ per molecule with an association constant of about 3 X 10(3) M-1. Apo-alpha-lactalbumin binds one Cu2+ per molecule with association constant 8 X 10(4) M-1 and from two to three Cu2+ with an association constant of about 4 X 10(3) M-1. The results obtained from spectrofluorometric pH titration of alpha-lactalbumin in the acidic pH region show the possible involvement of histidine residues in the coordination of Cu2+. The binding of Cu2+ to alpha-lactalbumin lowers significantly its thermostability and stability towards urea denaturation. The stability of Cu2+, Ca2+-alpha-lactalbumin against thermal and urea denaturation is similar to that of the apo protein. The thermal transition in Cu2+, Ca2+-alpha-lactalbumin occurs within the region of physiological temperatures which may suggest the existence of some thermal regulation of its functioning in vivo.

Binding Sites↗

PubMed1988

Interactions of calcium binding proteins, parvalbumin and alpha-lactalbumin, with dipalmitoylphosphatidylcholine vesicles.

Interactions of Ca2+ binding proteins, pike (Esox lucius) parvalbumins pI 4.2 and 5.0, and bovine and human alpha-lactalbumins, with dipalmitoylphosphatidylcholine vesicles were studied by means of scanning microcalorimetry and intrinsic tyrosine and tryptophan fluorescence methods. The interactions of pike parvalbumins are modulated by Ca2+ and Mg2+ binding to the protein and induce some changes in the physical properties of both the proteins and liposomes. Liposomes increased thermal stability of Ca2+-loaded parvalbumin and decreased thermal stability of both Mg2+-loaded and metal-free protein. The interaction of parvalbumin with liposomes affects the phase transition from gel to liquid-crystalline state in liposomes. Ca2+-loaded alpha-lactalbumin interacts with liposomes in its native state while the metal-free protein binds to the liposomes mainly in its thermally denatured state. The results of the microcalorimetric and spectrofluorometric studies are supported by data obtained by means of gel-chromatography on Sepharose 4B. It may be suggested that these metal-modulated interactions of Ca2+-binding proteins with membranes have some functional significance.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1987

On experimental artifacts in the use of metal ion chelators for the determination of the cation binding constants of alpha-lactalbumin. A reply.

The binding constant of Ca2+ to the strong cation site of bovine alpha-lactalbumin has been measured directly by monitoring the free calcium concentration by Quin 2 fluorescence. A dissociation constant of 1-4 nM was calculated, which confirms the strong calcium binding properties of this protein. In order to examine whether the metal ion chelators EDTA or EGTA affect the cation binding equilbria by binding to bovine alpha-lactalbumin, calcium binding equilibria were carefully measured under highly stabilized pH and temperature conditions. Within the concentration ranges required for competitive binding by these ligands (EDTA or EGTA) (less than 1-3 mM) these chelators produced no artifacts, in contradiction to the data of Kronman and Bratcher (Kronman, M. J., and Bratcher, S. C. (1983) J. Biol. Chem. 258, 5707-5709).

Binding, Competitive↗

PubMed1987

Stopped-flow kinetic studies of Ca(II) and Mg(II) dissociation in cod parvalbumin and bovine alpha-lactalbumin.

The dissociation kinetics of complexes of bovine alpha-lactalbumin and cod parvalbumin with Ca(II) and Mg(II) ions induced by mixing of a Ca(II)- or MG(II)-loaded protein with a chelator of divalent cations (EDTA or EGTA) have been studied by means of the stopped-flow method with intrinsic protein fluorescence registration. Within the temperature interval from 10 to approx. 37 degrees C kinetic curves for Ca(II) removal from alpha-lactalbumin are monoexponential with a rate constant ranging from 0.006 to 1 s. Taking into account the rather low rate of fluorescence changes, one can assume that the limiting stage in this case is the dissociation of the single bound Ca(II) ion from the protein and not a conformational transition which occurs after Ca(II) dissociation. At temperatures above 37 degrees C the kinetic curves require at least two exponential terms for a satisfactory fit. The second exponential seems to be due to denaturation of the apo form of alpha-lactalbumin which takes place at these temperatures. The values of the dissociation rate constants for Mg(II) bound to alpha-lactalbumin practically coincide with those for Ca(II). Within the temperature interval 10-30 degrees C the kinetic curves for Ca(II) and Mg(II) removal from parvalbumin are best fitted by a sum of two exponential terms identified as arising from the dissociation of cations from the two binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Effects of pH, temperature and Ca2+ content on the conformation of alpha-lactalbumin in a medium modelling physiological conditions.

Data obtained by the intrinsic protein fluorescence technique showed that, in addition to Ca2+ and Mg2+ ions, bovine alpha-lactalbumin also binds physiologically significant Na+ and K+ ions, the nucleotides ATP, ADP, UTP, UDP and UDP-galactose. The release of the bound Ca2+ ions from the protein in a medium modelling physiological conditions (containing Mg2+, Na+, K+, ATP and ADP in physiological concentrations) induced a transition of the protein from the native state of the Ca2+-loaded form to a state which is a mixture of native and and thermally changed states of the apo- and metal bound forms. Any variations in temperature result in changes in the populations of these states. This may be associated with some Ca2+ and temperature dependent regulation of the protein function. Variations of pH within the physiological limits had little influence on the conformation of both Ca2+-loaded and Ca2+-free alpha-lactalbumin.

Adenosine Diphosphate↗

PubMed1986

Thermal transitions in rat erythrocyte ghosts.

Thermal transitions in rat erythrocyte ghosts have been studied by means of scanning microcalorimetry, intrinsic fluorescence, circular dichroism and infrared spectroscopy. Heating of the suspension of rat erythrocyte ghosts induced at least four thermodynamically irreversible transitions. Each of these transitions is believed to be due to a localized structural transition induced by thermal stress. The influence of pH, papain, phospholipase and other modifications on the thermal transitions in rat erythrocyte ghosts is demonstrated.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗