Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.
- MeSH
- bromkyan metabolismus MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- cystein metabolismus MeSH
- dimerizace MeSH
- disulfidy metabolismus MeSH
- endopeptidasy chemie metabolismus ultrastruktura MeSH
- fluorescenční spektrometrie MeSH
- genové produkty gag metabolismus MeSH
- kinetika MeSH
- Masonův-Pfizerův opičí virus enzymologie fyziologie MeSH
- molekulární sekvence - údaje MeSH
- molekulová hmotnost MeSH
- mutantní proteiny chemie metabolismus MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- posttranslační úpravy proteinů MeSH
- replikace viru fyziologie MeSH
- retrovirové infekce MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- stabilita enzymů MeSH
- termodynamika MeSH
- virion fyziologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bromkyan MeSH
- cystein MeSH
- disulfidy MeSH
- endopeptidasy MeSH
- genové produkty gag MeSH
- Mason-Pfizer monkey virus protease MeSH Prohlížeč
- mutantní proteiny MeSH
Electromigration capillary methods are promising techniques in proteomics and they are still under research. We used a partial filling approach, i.e. a combination of gel and non-gel separation mechanisms in a single dimension. We tried using an interesting gel, Pluronic F 127, which can be considered as a surfactant capable of self-association both with isotropic and anisotropic gels. The Pluronic was inserted inside the capillary as a plug at the start of the capillary, and it provided separation at the first time. Separation by this gel was achieved according to molecular weight and/or hydrophobicity. The applicability of this method was demonstrated in the separation of real samples-peptides arising from collagen after CNBr or collagenase cleavage and albumin after trypsin cleavage (peptide mapping). Some peptides and proteins were selectively retained by the Pluronic gel. These interactions with the gel did not depended on their molecular weight alone, but they probably depend on a combination of both principles. It was confirmed that capillary electrophoresis with Pluronic plug can give us another new separation option, complementary to free solution capillary electrophoresis. The CE method presented here, consisting of a partial filling approach with combine gel and non-gel separation mechanisms seemed to be a promising method for the separation of complex mixtures of peptides.
- MeSH
- bromkyan chemie MeSH
- elektroforéza kapilární přístrojové vybavení metody MeSH
- gely chemie MeSH
- kolagenasy metabolismus MeSH
- krysa rodu Rattus MeSH
- peptidy analýza chemie izolace a purifikace MeSH
- poloxamer chemie MeSH
- proteiny analýza chemie izolace a purifikace MeSH
- sérový albumin hovězí analýza chemie MeSH
- skot MeSH
- trypsin metabolismus MeSH
- ultrafiltrace MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bromkyan MeSH
- gely MeSH
- kolagenasy MeSH
- peptidy MeSH
- poloxamer MeSH
- proteiny MeSH
- sérový albumin hovězí MeSH
- trypsin MeSH
Several new biocompatible and degradable materials were prepared by chemical modification of sodium hyaluronate. The method of activation of hyaluronate by cyanogen bromide was used and subsequent reaction with nucleophile led to the formation of carbamate. This modification of hydroxyl groups of glycosaminoglycans preserves the carboxyl groups and retains properties of polyelectrolyte. This method affords derivatives easily and the reaction condition correlates with degree of substitution. The experimental results show the effect of reaction conditions (reaction time, ratio of reactants) and effect of substitution on biodegradability. The obtained materials were characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy.
- MeSH
- biodegradace MeSH
- biokompatibilní materiály chemická syntéza MeSH
- bromkyan chemie MeSH
- gelová chromatografie MeSH
- glykosaminoglykany chemie MeSH
- hyaluronoglukosaminidasa farmakologie MeSH
- hydroxylový radikál chemie MeSH
- karbamáty chemická syntéza MeSH
- kinetika MeSH
- kyselina hyaluronová chemická syntéza chemie MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- rozpustnost MeSH
- skot MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- viskozita MeSH
- voda chemie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- biokompatibilní materiály MeSH
- bromkyan MeSH
- glykosaminoglykany MeSH
- hyaluronoglukosaminidasa MeSH
- hydroxylový radikál MeSH
- karbamáty MeSH
- kyselina hyaluronová MeSH
- voda MeSH
Heptanesulfonic acid as ion-pairing agent was used for the separation of mixtures of low and high molecular mass peptides/proteins by capillary electrophoresis. The separation conditions used were: capillary 37 cm (30 cm to the detector) x 75 microm i.d., voltage 10 kV, phosphate buffer 50 mmol/l, ion-pairing agent heptanesulfonic acid at three different concentrations, namely, 0, 20 or 100 mmol/l, pH 2.5. The separation reflected the ion-pairing equilibria between the ion-pairing agent and the peptide/protein analytes. The influence of ion-pairing on sample mobility (running time) was more pronounced in case of the higher-molecular peptides as compared to the low molecular ones. This difference offers the possibility to separate low and high molecular peptides/proteins that under the absence of the ion-pairing agent would co-migrate. The principle of this approach was demonstrated on a randomly selected set of peptides/proteins; the practical applicability was demonstrated on a set of CNBr peptides arising from a naturally occurring mixture of collagen types I and III.
- MeSH
- bromkyan MeSH
- cytochromy c chemie MeSH
- elektroforéza kapilární MeSH
- hydrolýza MeSH
- indikátory a reagencie MeSH
- kolagen chemie MeSH
- krysa rodu Rattus MeSH
- kyseliny sulfonové chemie MeSH
- molekulová hmotnost MeSH
- peptidy chemie izolace a purifikace MeSH
- proteiny chemie izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bromkyan MeSH
- cytochromy c MeSH
- indikátory a reagencie MeSH
- kolagen MeSH
- kyseliny sulfonové MeSH
- peptidy MeSH
- proteiny MeSH
A number of electromigration separation modes were applied to the separation of CNBr-released peptides from rat tail tendon collagen (microemulsion electrokinetic chromatography, methanol- or ethanol-modified background electrolytes and the separation in the presence of molecular sieving effect exerting polymer, both in the presence and absence of SDS). Electrodriven separations with a Hypersil C8 packed capillary were investigated as well. The best separations were obtained with either the molecular sieving effect exerting polymer (polyethylene oxide) in the background electrolyte (whether SDS was present or absent) or with the electrodriven chromatography using the C8 reversed-phase packed capillary. In the latter separation system, it was possible to separate 25-27 peaks of the theoretically expected 24 peptides in the analyzed mixture of which 17 were at least tentatively identified. The additional peaks apparently stem from the incomplete cleavage of the parent collagen alpha chains. Successful separations can be done either with predominating molecular sieving or hydrophobic partitioning mechanism.
- MeSH
- bromkyan chemie MeSH
- chemické modely MeSH
- chromatografie micelární elektrokinetická kapilární metody MeSH
- kolagen chemie MeSH
- peptidové fragmenty chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- bromkyan MeSH
- kolagen MeSH
- peptidové fragmenty MeSH
A capillary electrophoretic method exploiting the properties of Pluronic copolymer liquid crystals (F127) was developed for the separation of collagen cyanogen bromide (CNBr) fragments. The separations obtained were at least comparable (if not better) to those obtained by other methods applicable to this category of compounds. In the optimized version a bare silica capillary [47 cm (40 cm to the detector) x 75 microm I.D.] was used with 10 mM Tris and 75 mM phosphate buffer (pH 2.5) containing 7.5% Pluronic F127 copolymer. The separation mechanism which involves both the molecular sieving and surfactant properties of the Pluronic F127 gel phase is discussed.
- MeSH
- bromkyan chemie izolace a purifikace MeSH
- elektroforéza kapilární metody MeSH
- kolagen izolace a purifikace MeSH
- krysa rodu Rattus MeSH
- peptidové fragmenty izolace a purifikace MeSH
- polyethyleny chemie MeSH
- polypropyleny chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- bromkyan MeSH
- kolagen MeSH
- peptidové fragmenty MeSH
- polyethyleny MeSH
- polypropyleny MeSH
- UCON 50-HB-5100 MeSH Prohlížeč
Current techniques used for collagen alpha-chains and their CNBr fragments are reviewed. Ion exchange, gel permeation, reversed-phase and affinity chromatography are discussed mainly from the preparative aspects as these are both the techniques of choice to remove biological matrix contaminants always present in collagen preparations and techniques routinely used for preparative purposes. Among electromigration procedures gel electrophoresis is widely used both for intact collagen alpha-chains and their fragments. Recently this technique was applied also for miniaturised preparations. Immunoblotting techniques serve more specific detection of otherwise hard to distinguish different collagen polypeptide chains. Capillary electromigration techniques brought recently new aspects of understanding the behaviour of collagen proteins upon different separation modes and seem to represent a smart perspective for better quantitation of individual collagen species.
- MeSH
- bromkyan chemie MeSH
- elektroforéza metody MeSH
- kolagen izolace a purifikace MeSH
- lidé MeSH
- peptidové fragmenty izolace a purifikace MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- bromkyan MeSH
- kolagen MeSH
- peptidové fragmenty MeSH
Binding of lead (as lead acetate) to collagen type I alpha, and alpha2 chains, collagen type V and a large cyanogen bromide fragment of type I collagen [alpha2(I)CB(3,5)] was investigated by the large-zone Hummel-Dreyer method. It was demonstrated that two categories of binding sites exist in the collagen molecule, the number of which correlates rather well with the available aspartic and glutamic acid residues. Similar results were obtained for all collagen chains (fragments) used. The number of sites thus obtained was compared with the cross-striation pattern (reflecting areas where lead is bound) of the SLS form of collagen type I (alpha1 chain); it is suggested that the number of bands seen in the SLS form reflects primarily the number of available aspartic acid residues in the molecule. The association constants obtained are comparable with the low affinity interactions seen e.g., between Cu and bovine serum albumin.
Capillary electrophoresis separation and synchronous fluorescence spectral detection was used off-line to reveal the nature of fluorescent adducts formed in vivo in the collagen molecule and their distribution in the molecule. It was shown that by using the delta lamda in the area of the Stokes shift for the analyzed entities (approximately 10 nm for pentosidine, 4,5(E)-epoxy-2(E)-heptenal and 4,5(E)-epoxy-2(E)-decenal lysine adducts) a distinct profile of spectral bands can be obtained allowing for differentiation of the several entities involved. In combination with capillary electrophoretic separation of the CNBr peptides the location of individual adducts was possible: while pentosidine (and, perhaps, pentosidine related compounds K1-K4) is found in the large alpha 1(I)CB6 and alpha 2(I)CB3.5 peptides along with a complete set of the other fluorescent adducts, low-molecular-mass peptides originating from the terminal region of the molecule are devoid of any fluorescence. All other parts of the molecule possess synchronous fluorescence profiles corresponding to the intact molecule except that they are devoid of pentosidine. The results indicate random distribution of fluorescent adducts in the collagen molecule and, in a broader context, indicate the usefulness of multicomponent analysis by means of combining synchronous luminescence spectra and capillary electrophoresis.
- MeSH
- algoritmy MeSH
- bromkyan MeSH
- elektroforéza kapilární MeSH
- fluorescenční barviva MeSH
- fluorescenční spektrometrie MeSH
- glykosylace MeSH
- kolagen analýza MeSH
- krysa rodu Rattus MeSH
- kůže chemie MeSH
- luminiscenční měření MeSH
- peptidy analýza MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bromkyan MeSH
- fluorescenční barviva MeSH
- kolagen MeSH
- peptidy MeSH
Combination of standard approaches like pepsin digestion and slab gel electrophoresis with capillary separations allows a relatively easy identification of in vivo occurring collagen fragments. Capillary electrophoresis can be done either in 25 mM phosphate buffer (pH 2.5) or in a 25 mM phosphate buffer (pH 4.5) made 0.1% with respect to sodium dodecyl sulfate (SDS). While in the first case peptides move to the cathode in a molecular mass dependent manner, in the second case they move towards anode (also in a molecular mass dependent manner). The profiles obtained by the two approaches resemble mirror images with low molecular mass peptides moving first in the acid background electrolyte while they move last in the presence of SDS. It is proposed that in the capillary electrophoretic separation at pH 2.5 the separation mechanism involves the interaction of the individual peptides with the capillary wall while in the second case (pH 4.5) the leading mechanism of separation involves the interaction of the analytes with the micellar phase. For micellar phase separation the system must be run at reversed polarity. Capillary electrophoretic separation in the pH 2.5 buffer is considerably affected by the presence of SDS in the previous steps of peptide preparation. If the peptides are obtained from SDS slab gel electrophoresis, their movement in the capillary electrophoresis step is about three times faster that the movement of corresponding peptides which have not been complexed with SDS.
- MeSH
- arteria pulmonalis chemie MeSH
- bromkyan chemie MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- hypoxie metabolismus MeSH
- imunoblotting MeSH
- kolagen analýza chemie metabolismus MeSH
- kolagenasy metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- krysa rodu Rattus MeSH
- peptidové fragmenty analýza chemie MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- bromkyan MeSH
- kolagen MeSH
- kolagenasy MeSH
- peptidové fragmenty MeSH
