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Biomedical subjects

G Kern

Publications and source records attributed to G Kern.

At least 19 recordsLinked to original sources
PubMed2002

Mechanisms of acid secretion in pseudobranch cells of rainbow trout (Oncorhynchus mykiss).

Cell suspensions of rainbow trout Oncorhynchus mykiss pseudobranch, prepared by Ca(2+) depletion and mechanical maceration, contained a distinct population of cells that always kept their relatively cuboidal shape and did not round up in suspension or proliferate after adhering to the surface of cell culture dishes. Phasecontrast microscopy revealed an extensive system of basal membrane invaginations, and Na(+)-K(+)-ATPase- and anionexchanger-like immunoreactivity could be localized in cell membranes. The cells were characterized by a high mitochondrial density. Using specific antibodies, V-ATPase subunit B was localized in the plasma membrane. Using a cytosensor microphysiometer, the rate of acid secretion of these cells was measured and compared with the activity of a gill cell preparation. Incubation of pseudobranch cells with bafilomycin A1 (10(-6) mol l(-1)), a specific inhibitor of V-ATPase, reduced the rate of acid secretion by about 10% under control conditions, while no effect of bafilomycin on the rate of acid secretion of gill cells was observed. Application of amiloride (5 x 10(-5) mol l(-1)) reduced the rate of acid secretion in cells of both organs, pseudobranch and gills. Incubation of pseudobranch cells with DIDS (10(-3) mol l(-1)) resulted in a minor increase in the rate of proton secretion, but in cells prepared from the gills of rainbow trout acid secretion was reduced by about 30-40%. It is concluded that pseudobranch cells are equipped with various pathways to secrete protons, and that the anion exchange activity especially of pseudobranch cells appears to be different from that in gills.

Animals↗
PubMed1998

Activation of thiamin diphosphate in enzymes.

Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

Allosteric Regulation↗
PubMed1998

Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum.

The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10(-2) s-1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s-1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in pyruvate oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-pyruvate oxidase ternary complex requires a second step, which was detected at low temperature.

Catalysis↗
PubMed1998

Characterization of a folding intermediate from HIV-1 ribonuclease H.

The RNase H domain from HIV-1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped-flow far UV circular dichroism and pulse-labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog from Escherichia coli, the rate limiting step in refolding of HIV RNase H appears closer to the native state. We have modeled this kinetic intermediate using a C-terminal deletion fragment lacking helix E. Like the kinetic intermediate, this variant folds rapidly and shows a decrease in stability. We propose that inhibition of the docking of helix E to this folding intermediate may present a novel strategy for anti HIV-1 therapy.

Circular Dichroism↗
PubMed1997

Molecular mechanism of regulation of the pyruvate dehydrogenase complex from E. coli.

The pyruvate dehydrogenase multienzyme complex from E. coli shows a sigmoidal dependency of the reaction rate on the substrate concentration when product formation is followed in the presence of physiological concentrations of the cofactor thiamin diphosphate. To elucidate the molecular mechanism of this regulation, the influence of the substrate pyruvate on the coenzyme-protein interaction has been investigated using several coenzyme analogues. The observed binding constants of all coenzymatically active analogues are increased in the presence of the substrate pyruvate, whereas those of all coenzymatically inactive analogues are not altered in the presence of pyruvate. This points to an increased binding affinity of a reaction-intermediate-coenzyme complex to the protein. Since cofactor binding and dissociation at physiological concentrations of thiamin diphosphate are slow compared to the catalytic reaction, a slow transition to the active state of the enzyme occurs. After lowering the pyruvate concentration, the opposite effect, a dissociation of the thiamin diphosphate from the enzyme is observed. This slow substrate dependent enhancement of cofactor binding enables efficient regulation of the pyruvate dehydrogenase complex by its substrate pyruvate.

Coenzymes↗
PubMed1997

How thiamine diphosphate is activated in enzymes.

The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

Allosteric Regulation↗
PubMed1996

Autocatalytic folding of the folding catalyst FKBP12.

Prolyl isomerases are folding enzymes and thus have the potential to catalyze their own folding. We show here that the folding of cytosolic FKBP12 (FK 506 binding protein) is an autocatalytic process both for the mature protein and for a fusion protein with an amino-terminal extension of 16 residues. Native FKBP contains seven trans-prolyl peptide bonds, and the cis-to-trans isomerizations of some or all of them constitute the slow, rate-limiting events in folding. The rate of an autocatalytic reaction increases with reactant concentration, because the product catalyzes its own formation. Accordingly, the folding of the fusion protein was more than 10-fold accelerated when the protein concentration was increased from 0.05 microM to 10 microM. At high concentrations of both forms of FKBP12 autocatalysis was very efficient, and the observed folding rate seemed to approach the rate of the fast direct folding reaction of the protein molecules with the correct (all trans) peptidyl-prolyl bond conformation.

Amino Acid Isomerases↗
PubMed1995

Kinetic analysis of cyclophilin-catalyzed prolyl cis/trans isomerization by dynamic NMR spectroscopy.

To investigate the kinetics of the prolyl peptide bond cis/trans isomerization of N-succinyl-Ala-Phe-Pro-Phe-(4)-nitroanilide catalyzed by peptidyl prolyl cis/trans isomerases (PPIases), one-dimensional dynamic 1H NMR spectroscopy was employed. To this end line shape analyses of proton signals were performed at various concentrations of both cytosolic porcine kidney cyclophilin (Cyp18) and peptide substrate. Catalysis of the cis/trans isomerization by Cyp18 is best described by a four-site exchange model, where the four sites represent the cis and trans isomers free in solution and bound to the enzyme. Combination of dynamic NMR spectroscopy with the classical protease-coupled PPIase assay allowed determination of the complete set of the microscopic rate constants describing the four site exchange model. The comparison of the rate constants of cis-->trans isomerization of the peptide free in solution and bound to cyclophilin yields an acceleration factor of 3.5 x 10(5). Dissociation of the Michaelis complexes are of the same order of magnitude as the isomerization rates on the enzyme. Therefore, all microscopic rate constants contribute to the steady state parameters. For the first time, the kcat (620 s-1) and KM (220 microM) value for the trans isomer in addition to the values of the cis isomer (kcat = 680 s-1, KM = 80 microM) could be determined under reversible conditions at pH 6.0 and 10 degrees C. The affinity of Cyp18 for the cis isomer is 4 times higher than for the trans isomer. This results in a shift of the cis/trans equilibrium toward the cis isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗
PubMed1995

A kinetic analysis of the folding of human carbonic anhydrase II and its catalysis by cyclophilin.

The kinetics of unfolding and refolding of human carbonic anhydrase II (HCAII) and its catalysis by the peptidyl-prolyl-cis/trans-isomerase cyclophilin were investigated. HCAII contains 15 trans- and 2 cis-prolyl peptide bonds, and, when long-term denatured, virtually all unfolded molecules contain non-native prolyl isomers. In unfolding these molecules (Us) are produced slowly in a biphasic process reflecting the isomerization of several trans-prolines and of one cis-proline. In refolding, the rapid formation of an intermediate of the molten globule type is followed by several slow prolyl isomerizations, which determine the rate of reactivation. By a short 10-s incubation in 5.0 M guanidinium chloride at 2 degrees C, unfolded HCAII species with all prolines still in the native conformation (Uf) could be produced. Surprisingly, only a fraction of Uf refolds rapidly, but the other molecules refold slowly. Evidently, some prolyl peptide bonds isomerize early in refolding, at the stage of the molten globule and as a consequence, molecules with incorrect prolyl isomers are formed in competition with the productive folding of Uf. This fraction of slow-folding molecules is strongly increased when cyclophilin is present, because it accelerates the formation of non-native prolyl isomers as long as the molecules remain in the molten globule state. Later cyclophilin catalyzes the isomerization of these prolyl peptide bonds toward the native state, which are stabilized in their conformation by further folding to the native state. This catalysis is very efficient, because only prolines that are accessible in the molten globule are involved in this sequence of isomerization and reisomerization.

Amino Acid Isomerases↗
PubMed1994

Reassessment of the putative chaperone function of prolyl-cis/trans-isomerases.

The folding of proteins can be assisted by two unrelated groups of helper molecules. Chaperones suppress non-productive side reactions by stoichiometric binding to folding intermediates, and folding enzymes catalyze slow rate-limiting steps of folding. We reinvestigated, whether peptidyl-prolyl-cis/trans-isomerases of the cyclophilin type act simultaneously as chaperones and as folding catalysts in the reactivation of human carbonic anhydrase II, as reported recently [Freskgård, P.-O. et al. (1992) Science 258, 466-468; Rinfret, A. et al. (1994) Biochemistry 33, 1668-1673]. No increase in the yield of native carbonic anhydrase-II could be detected in the presence of three different prolyl isomerases, when reactivation was followed by a sensitive assay for an extended time of 4 h. We conclude that the role of prolyl isomerases in the refolding of carbonic anhydrase can be explained solely by their isomerase activity. There is no need to invoke simultaneous functions as chaperones for these folding catalysts.

Amino Acid Isomerases↗
PubMed1993

Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae.

A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus. This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo. In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene. Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions. The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol. The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type. Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography. The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme Reactivators↗
PubMed1992

Glycosylation inhibits the interaction of invertase with the chaperone GroEL.

During refolding and reassociation of chemically denatured non-glycosylated invertase from Saccharomyces cerevisiae, aggregation competes with correct folding, leading to low yields of reactivation (Kern et al. (1992) Protein Sci. 1, 120-131). In the presence of the chaperone GroEL, refolding is completely arrested. This suggests the formation of a stable complex between GroEL and non-native non-glycosylated invertase. Addition of MgATP results in a slow release of active invertase from the chaperone complex. When GroEL/ES and MgATP are present during refolding, the final reactivation yield increases from 14% to 36%. In contrast, refolding of the core-glycosylated and the high-mannose glycosylated forms of invertase is not arrested by GroEL. Only a short lag phase at the beginning of reactivation and a slightly increased reactivation yield (64% to 86% for core-glycosylated and 62% to 76% for external invertase) indicate a weak interaction of the glycosylated forms with the chaperone.

Bacterial Proteins↗
PubMed1992

Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.

Enzyme Reactivators↗
PubMed1988

Effect of DNA sequence and structure on nuclease activity of the DexA protein of bacteriophage T4.

The bacteriophage T4 dexA gene product is required during infection of Escherichia coli strains carrying a mutation in the optA gene. We purified the DexA protein from cells which overproduced the protein. The protein was assayed for nuclease activity on synthetic di- and oligonucleotide substrates of known sequence and secondary structure. Sequence and structure significantly affected nuclease activity. The properties of the enzyme may explain the requirement for the DexA protein during infection of optA mutant hosts.

Base Sequence↗
PubMed1986

[Hepatitis A outbreak in a shelter for the homeless in Vienna].

An outbreak of hepatitis A was observed in a shelter for the homeless in Vienna with about 200 inhabitants. Twenty-two cases occurred within a period of 6 months. The outbreak could not be brought under control by measures of general hygiene. However, after the administration of hepatitis A immunoglobulin (120 IU/ml), at a dosage of 0.05 ml/kg body weight, to 102 of the 105 seronegative inhabitants and members of staff, no further clinical cases of hepatitis A were reported from this group. Nevertheless, 8 of these 102 "protected" persons showed signs of subclinical infection at subsequent follow up. Apart from these, 2 further cases of hepatitis A occurred among the non-immunised children at risk, whose parents had refused permission for serological investigation or immunoglobulin administration.

Adolescent↗
PubMed1986

Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay, indirect immunofluorescence, and virus isolation: a comparative study.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of respiratory syncytial virus (RSV) antigens in nasopharyngeal secretions (NPS) from children with acute respiratory disease. Antisera against RSV nucleocapsids were used as immunoreagents for this test system. The results obtained by RSV antigen ELISA were compared to those of indirect immunofluorescence (IF) and tissue culture virus isolation (TC). Of the 404 NPS obtained, 278 were tested in parallel by ELISA and IF and 205 by ELISA and TC, and 89 were screened in parallel by all three methods. The sensitivity of ELISA in relation to IF was 86.7%, the specificity 95.7%. Sensitivity and specificity obtained by ELISA were 89.9% and 94.4%, respectively, compared to TC. False-negative results were obtained with all three test systems used.

Adolescent↗
PubMed1986

[Management of mammillary discharge. Apropos of 38 cases].

The authors offer a diagnostic approach in cases of mammillary discharge and present a study of 38 cases of spontaneous discharge. The diagnosis is based on clinical findings combined with supplementary examinations, beginning with a cytological examination of the discharges, either bilateral or, in most cases, unilateral. The supplementary investigations are standard: mammography, galactography, with more and more frequent recourse to ultrasonography. If warranted on sufficient grounds, biopsy by sectorectomy is carried out to provide a histological diagnosis. The observed results show epithelial vegetation with (38%) and without (37.5%) atypical cells, the histological interpretation of which is: benign papilloma: 50 percent, diffuse papillomatosis: 17 percent, galactophoric carcinoma: 1 case and galactophoric ectasias: 22 percent. This study is compared to the results of Mouriquand et al [10] on induced discharges, where the frequency of papilloma and papillomatosis is slightly inferior by about 5 percent.

Biopsy↗