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M Linder

Publications and source records attributed to M Linder.

At least 55 records · Page 3Linked to original sources
PubMed1998

Solution structure of the cellulose-binding domain of endoglucanase I from Trichoderma reesei and its interaction with cello-oligosaccharides.

The solution structure of a synthetic 38-residue cellulose-binding domain (CBD) of endoglucanase I from Trichoderma reesei (CBD(EGI)) was determined by two-dimensional 1H-NMR spectroscopy. 100 structures were generated from a total of 599 NOE derived distance restraints and 28 phi and 14 chi dihedral angle restraints. For the final set of 19 selected structures, the rms deviation about the mean structure was 0.83+/-0.26 A for all atoms and 0.50+/-0.22 A for the backbone atoms. The structure of CBD(EGI) was very similar to that of CBD of cellobiohydrolase I from T reesei (CBD(CBHI)). The backbone trace of CBD(EGI) followed closely the irregular triple-stranded antiparallel beta-sheet structure of CBD(CBHI). Moreover, apart from the different side chains of Trp7 (CBD(EGI)) and Tyr5 (CBD(CBHI)), the cellulose-binding face of CBD(EGI) was similar to that of CBD(CBHI) within the precision of the structures. Finally, the interaction between CBD(EGI) and soluble sugars was investigated using cellopentaose and cellohexaose as substrates. Experiments showed that the interactions between CBD(EGI) and cellobiose units of sugars are specific, supporting the previously presented model for the CBD binding to crystalline cellulose.

Amino Acid Sequence↗
PubMed1998

A specific binding protein for cardiac glycosides exists in bovine serum.

Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-+ ++aminocapro ate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation. The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with Kd' of 1.52 nM and Kd' = 75 nM and with an interaction factor of 50 using a Koshland-Némethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals.

Affinity Labels↗
PubMed1998

Structure and glycosylation patterns of surface proteins from woodchuck hepatitis virus.

Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.

Amino Acid Sequence↗
PubMed1998

A history of medical scientists on high heels.

For 250 years medical scientists have propagandized about the health hazards of high-heeled shoes, which originated four centuries ago. Physicians, however, largely unaware of their own profession's tradition, keep reinventing the diagnostic wheel. This professional amnesia has held back the momentum of the process of educating the public. Consequently, despite these warnings, millions of women continue to wear high-heeled shoes. This article describes the history of the medical profession's recognition of this worldwide health problem and the current understanding of the deleterious and often irreversible biomechanical effects of high-heeled shoewear. The article emphasizes that the reemergence of high heels and of medical interest in them in the third quarter of the 19th century, following their disappearance in the wake of the French Revolution, was associated with increasing pressure by employers to wear such shoes for long hours at work. Although medical scientists have recognized this specifically occupational phenomenon for more than a century, full-scale epidemiological studies may be necessary to bring about substantial social-behavioral change.

Biomechanical Phenomena↗
PubMed1997

Trichoderma reesei cellobiohydrolase I with an endoglucanase cellulose-binding domain: action on bacterial microcrystalline cellulose.

Cellulolytic enzymes consist of distinct catalytic and cellulose-binding domains (CBDs). The presence of a CBD improves the binding and activity of cellulases on insoluble substrates but has no influence on their activities on soluble substrates. Structural and biochemical studies of a fungal CBD from Trichoderma reesei cellobiohydrolase I have revealed a wedge shaped structure with a flat cellulose binding surface containing three essential tyrosine residues. The face of the wedge is strictly conserved in all fungal CBDs while many differences occur on the other face of the wedge. Here we have studied the importance of these differences on the function of the T. reesei CBHI by replacing its CBD by a homologous CBD from the endoglucanase, EGI. Our data shows that, apart from slightly improved affinity of the hybrid enzyme, the domain exchange does not significantly influence the function of CBHI.

Amino Acid Sequence↗
PubMed1997

Interaction between cellohexaose and cellulose binding domains from Trichoderma reesei cellulases.

Most Trichoderma reesei cellulases consist of a catalytic and a cellulose binding domain (CBD) joined by a linker. We have used cellohexaose as a model compound for the glucose chain to investigate the interaction between the soluble enzyme and cellulose. The binding of cellohexaose to family I CBDs was studied by NMR spectroscopy. CBDs cause line broadening effects and decreasing T2 relaxation times for certain cellohexaose resonances, whereas there are no effects in the presence of a mutant which binds weakly to cellulose. Yet it remains uncertain how well the soluble cellooligosaccharide mimics the binding of CBD to the cellulose.

Binding Sites↗
PubMed1997

Analyses of disulfides present in the rubella virus E1 glycoprotein.

The surface of Rubella virus contains the glycoproteins E1 and E2. The E1 protein induces neutralizing antibodies and has been implicated in the process of recognition of cellular receptors. To gain information on the structural organization of the E1 protein we have analyzed the disulfide bonds present within this molecule. The reactivity of the protein with radioactively labeled iodoacetic acid indicates that all 20 cysteine residues present in the ectodomain of the E1 protein are involved in disulfide formation. E1 protein was purified by preparative SDS-PAGE under nonreducing conditions from virus particles grown in tissue culture in the presence of [35S]cysteine. The purified protein was digested with a number of proteases followed by reversed phase high-performance liquid chromatography (HPLC). [35S]cysteine-containing peptides were identified and characterized by N-terminal amino acid sequence determination. These analyses identified the following eight disulfide bridges: C(1)-C(2); C(3)-C(15); C(6)-C(7); C(9)-C(10); C(11)-C(12); C(13)-C(14); C(17)-C(18); and C(19)-C(20). The two disulfide bridges formed by the residues C(4), C(5), C(8), and C(16) have not been identified with certainty, but a likely organization can be derived. The data obtained are discussed in the context of a possible structural and functional organization of the E1 protein.

Amino Acid Sequence↗
PubMed1997

Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei.

Three-dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel beta-sheet structure of the CBD fold was preserved in all engineered peptides. The three-dimensional NMR-based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N-terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.

Cellulase↗
PubMed1997

Thirst at work--an occupational hazard?

Of 1492 teachers 791 (53%) responded to a survey addressing whether voiding habits at work or behavioral factors influenced by this occupation predisposed women to urinary tract infection. The mean number of voids during the work day was 2.7 +/- 1.4; 24.5% voided infrequently (never or only once) and 26.5% voided four or more times during the work day; 15.8% had had a urinary tract infection in the preceding year. Half of the respondents made a conscious effort to drink less while working, to avoid needing to use the toilet. There was no association between the prevalence of urinary tract infection and the number of voids or infrequent voiding at work. Compared to women who drank the volume they desired at work, those who drank less had a 2.21-fold higher risk (95% CI 1.45-3.38) of urinary tract infection after controlling for being parous, voiding infrequently at work, and urge incontinence. Further study is warranted to determine whether modification of behavioral factors at work can reduce the incidence of urinary tract infections. If this association holds, public policy must be changed to allow workers more adequate access to toilet facilities.

Adult↗
PubMed1996

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.

Amino Acid Sequence↗
PubMed1996

Characterization of a double cellulose-binding domain. Synergistic high affinity binding to crystalline cellulose.

Most cellulose-degrading enzymes have a two-domain structure that consists of a catalytic and a cellulose-binding domain (CBD) connected by a linker region. The linkage and the interactions of the two domains represent one of the key questions for the understanding of the function of these enzymes. The CBDs of fungal cellulases are small peptides folding into a rigid, disulfide-stabilized structure that has a distinct cellulose binding face. Here we describe properties of a recombinant double CBD, constructed by fusing the CBDs of two Trichoderma reesei cellobiohydrolases via a linker peptide similar to the natural cellulase linkers. After expression in Escherichia coli, the protein was purified from the culture medium by reversed phase chromatography and the individual domains obtained by trypsin digestion. Binding of the double CBD and its single CBD components was investigated on different types of cellulose substrates as well as chitin. Under saturating conditions, nearly 20 micromol/g of the double CBD was bound onto microcrystalline cellulose. The double CBD exhibited much higher affinity on cellulose than either of the single CBDs, indicating an interplay between the two components. A two-step model is proposed to explain the binding behavior of the double CBD. A similar interplay between the domains in the native enzyme is suggested for its binding to cellulase.

Amino Acid Sequence↗
PubMed1996

Purification, properties and primary structure of H2-forming N5 ,N10 -methylenetetrahydromethanopterin dehydrogenase from Methanococcus thermolithotrophicus.

H2-Forming N5,N10 -methylenetetrahydromethanopterin dehydrogenase (Hmd) is a novel type of hydrogenase found in methanogenic Achaea that contains neither nickel nor iron-sulfur clusters. The enzyme has previously been characterized from Methanobacterium thermoautotrophicum and from Methanopyrus kandleri. We report here on the purification and properties of the enzyme from Methanococcus thermolithotrophicus. The hmd gene was cloned and sequenced. The results indicate that the enzyme from Mc. thermolithotrophicus is functionally and structurally closely related to the H2-forming methylene tetrahydromethanopterin dehydrogenase from Mb. thermoautotrophicum and Mp. kandleri. From amino acid sequence comparisons of the three enzymes, a phylogenetic tree was deduced that shows branching orders similar to those derived from sequence comparisons of the 16S rRNA of the orders Methanococcales, Methanobacteriales, and Methanopyrales.

Amino Acid Sequence↗
PubMed1995

The difference in affinity between two fungal cellulose-binding domains is dominated by a single amino acid substitution.

Cellulose-binding domains (CBDs) form distinct functional units of most cellulolytic enzymes. We have compared the cellulose-binding affinities of the CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from the fungus Trichoderma reesei. The CBD of EGI had significantly higher affinity than that of CBHI. Four variants of the CBHI CBD were made in order to identify the residues responsible for the increased affinity in EGI. Most of the difference could be ascribed to a replacement of a tyrosine by a tryptophan on the flat cellulose-binding face.

Adsorption↗