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J Markl

Publications and source records attributed to J Markl.

At least 37 records · Page 2Linked to original sources
PubMed1998

Zebrafish vimentin: molecular characterization, assembly properties and developmental expression.

To provide a basis for the investigation of the intermediate filament (IF) protein vimentin in one of the most promising experimental vertebrate systems, the zebrafish (Danio rerio), we have isolated a cDNA clone of high sequence identity to and with the characteristic features of human vimentin. Using this clone we produced recombinant zebrafish vimentin and studied its assembly behaviour. Unlike other vimentins, zebrafish vimentin formed unusually thick filaments when assembled at temperatures below 21 degrees C. At 37 degrees C few filaments were observed, which often also terminated in aggregated masses, indicating that its assembly was severely disturbed at this temperature. Between 21 and 34 degrees C apparently normal IFs were generated. By viscometry, the temperature optimum of assembly was determined to be around 28 degrees C. At this temperature, zebrafish vimentin partially rescued, in mixing experiments, the temperature-dependent assembly defect of trout vimentin. Therefore it is apparently able to "instruct" the misorganized trout vimentin such that it can enter normal IFs. This feature, that assembly is best at the normal body temperature of various species, puts more weight on the assumption that vimentin is vital for some aspects of generating functional adult tissues. Remarkably, like in most other vertebrates, zebrafish vimentin appears to be an abundant factor in the lens and the retina as well as transiently, during development, in various parts of the central and peripheral nervous system. Therefore, promising cell biological investigations may now be performed with cells involved in the generation of the vertebrate eye and brain, and, in particular, the retina. Moreover, the power of genetics of the zebrafish system may be employed to investigate functional properties of vimentin in vivo.

Amino Acid Sequence↗
PubMed1997

Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH).

Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain 1h, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5 mM MgCl2, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.

Amino Acid Sequence↗
PubMed1997

Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 A resolution by electron cryomicroscopy and angular reconstitution.

A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to approximately 45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the "functional units" within the dimeric "morphological units" of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.

Animals↗
PubMed1997

Keyhole limpet hemocyanin (KLH), II: Characteristic reassociation properties of purified KLH1 and KLH2.

Subunits of the two types of keyhole limpet hemocyanin (KLH1 and KLH2), purified by gel filtration chromatography and preparative polyacrylamide gel electrophoresis from Immucothel, have been used for macromolecular reassociation studies. In-vitro reassociation has been achieved with a standardized system using a Tris-saline stabilizing buffer at pH 7.4 containing 100 mM calcium and magnesium chloride at 4 degrees C. The relatively slow progress of reassociation has been monitored and the varying oligomeric forms of KLH1 and KLH2 produced have been studied by transmission electron microscopy, using specimens negatively stained with 5% ammonium molybdate containing 1% trehalose. Specimens have also been prepared by platinum-carbon shadowing, following freeze-cleavage. The two hemocyanins reassociate to produce characteristic oligomeric and polymeric forms. Subunits of purified KLH1 reassociate to produce a small number of didecamers, short multidecamers (ca 33 nm diameter) and much larger quantity of a ca 25 nm diameter flexible/undulatory tubular form of varying length. These tubules exhibit characteristic oblique features, indicative of an 'open' helical structure which appears to be a loosly or incompletely annealed twisted ribbon of subunits. After a period of days the tubules aggregate in parallel to produce large paracrystalline bundles, which do not have a tendency to associate end-to-end. Following transfer of this reassociated KLH1 to low calcium magnesium stabilizing buffer, the tubular bundles are unstable; they slowly break down into shorter lengths, fragments, subunit groups and individual subunits, which subsequently regenerate decamers, didecamers, and some multidecamers. Subunits of purified KLH2 reassociate to produce ca 25 nm diameter 'closed' tubules, which do not exhibit the oblique 'open' features shown by the KLH1 tubules; however, the ends of the these 'closed' tubules are often oblique. In addition to the tubular form, KLH2 reassociation also generates a somewhat small proportion of ca 33 nm diameter multidecamers, often containing many decamers and more than one 'nucleating' didecamer. On transfer to low calcium magnesium stabilizing buffer the KLH2 tubules are remarkably stable, but the number of multidecamers slowly increases with time. There is a significant structural difference between the short KLH1 multidecamers (only detected following in-vitro reassociation) and those of KLH2 quite apart from their length. Study of the metal shadowed specimens confirmed the difference between the KLH1 and KLH2 tubular forms, with relatively smooth helical surface ridges and a rougher internal surface, indicating internalization of subunit domains that are not required for the construction of the tubular wall, in accord with current understanding of the subunit organization within the native molecules.

Calcium Chloride↗
PubMed1997

Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2.

Studies of keyhole limpet hemocyanin (KLH) normally require purification of functional complexes directly from living animals. An alternative procedure is described wherein a commercial preparation of KLH which is fully dissociated into its subunits (Immucothel, biosyn Arzneimittel GmbH) is reassociated in the presence of a high concentration of calcium and magnesium. The reassociation products, when observed by electron microscopy, consist of didecamers, multidecamers and flexible tubules of varying length. The two forms of KLH described previously and designated KLH1 and KLH2, are present in the reassociated mixture as homo-oligomers/polymers and can be separated by selective dissociation of the KLH2 by treatment with 1% ammonium molybdate-0.2% PEG at pH 5.7, followed by gel filtration chromatography in this solution. In addition to discrete elution peaks containing didecameric KLH1 and dissociated subunits of KLH2, a leading peak contains a tubular/polymeric form of KLH1, not previously described. Under negative staining in conditions designed specifically for the creation of 2-dimensional crystals on mica (the negative staining-carbon film procedure), this tubular form of KLH1 can be transformed into a larger diameter multidecameric form, again not previously described for KLH1. The purified KLH2 peak is indistinguishable from subunit material prepared from living animals. This, Immucothel appears to provide a standardized source of subunits suitable for biochemical and structural studies on the two types of KLH.

Animals↗
PubMed1996

Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties.

We have isolated from a rainbow trout (Oncorhynchus mykiss) spleen cDNA library a clone coding for vimentin. The deduced amino acid sequence reveals a high degree of identity with vimentin from carp (81%), frog (71%), chick and human (73% each). Large stretches in the central alpha-helical rod are identical within all four classes of vertebrates, but in 17 residues spread over the entire rod, the two fish differ distinctly from the tetrapod species. In addition, in the more diverged non-helical head domain, a nonapeptide motif previously shown to be important for regular filament formation is conserved. Recombinant trout vimentin assembles into bona fide filaments in vitro, with a temperature optimum between 18 and 24 degrees C. Above 27 degrees C, however, filament assembly is abruptly abolished and short filaments with thickened ends as well as structures without typical intermediate filament appearance are formed. This distinguishes its assembly properties significantly from amphibian, avian and mammalian vimentin. Also in vivo, after cDNA transfection into vimentin-free mammalian epithelial cells, trout vimentin does not form typical intermediate filament arrays at 37 degrees C. At 28 degrees C, and even more pronounced at 22 degrees C, the vimentin-positive material in the transfected cells is reorganized in the perinuclear region with a partial fibrillar appearance, but typical intermediate filament arrays are not formed. Together with immunoblotting and immunolocalization data from trout tissues, where vimentin is predominantly found in glial and white blood cells, we conclude that vimentin is indeed important in its filamentous form in fish and other vertebrates, possibly fulfilling cellular functions not directly evident in gene targeting experiments carried out in mice.

Amino Acid Sequence↗
PubMed1994

Electron microscopy and biochemical characterization of a 350-kDa annular hemolymph protein from the keyhole limpet Megathura crenulata.

The isolation and biochemical characterization of an annular non-hemocyanin hemolymph protein from a marine gastropod, the Californian giant keyhole limpet (Megathura crenulata) is presented. By analytical ultracentrifugation, the protein has a sedimentation coefficient of 12S and molecular mass of approximately 350 kDa. The subunit mass, obtained by SDS/PAGE in the presence of -SH reagent and 8 M urea, is approximately 35 kDa, thereby indicating the presence of 10 subunits in the native molecule. By negative staining, the protein is revealed in one predominant image projection as a pentagonal approximately 8 nm ring-like structure with an approximately 2-nm stain-filled centre and, in another image projection, as a dimeric rectangular structure, approximately 8 nm x 14 nm. In deep stain, each of the components of the side-on dimer also shows an indication of stain penetration within a central channel. Unlike the hemolymph protein limulin/C-reactive protein from the horseshoe crab Limulus polyphemus, this Megathura protein does not possess any potential for the aggregation of phosphatidylcholine liposomes although it has an affinity for this phospholipid and causes bilayer destruction. It now appears likely that the previously reported lipid-bilayer ionic-conductance species in keyhole limpet hemolymph, attributed to hemocyanin, could indeed by due to the presence of this annular protein.

Animals↗
PubMed1992

Quaternary and subunit structure of Calliphora arylphorin as deduced from electron microscopy, electrophoresis, and sequence similarities with arthropod hemocyanin.

Arylphorin was purified from larvae of the blowfly Calliphora vicina and studied in its oligomeric form and after dissociation at pH 9.6 into native subunits. In accordance with earlier literature, it was electrophoretically shown to be a 500 kDa hexamer (1 x 6) consisting of 78 kDa polypeptides (= subunits). Electron micrographs of negatively stained hexamers show a characteristic curvilinear, equilateral triangle of 12 nm in diameter (top view) and a rectangle measuring 10 x 12 nm (side view). Alternatively, particles in the top view orientation exhibit a roughly circular shape 12 nm in diameter. Crossed immunoelectrophoresis revealed the presence of a major subunit type; the nature of a very minor and a third immunologically separated component remains unclear. A novel 2 x 6 arylphorin particle was detected and isolated. It comprises less than 10% of the total arylphorin material and shows a long, narrow interhexamer bridge in the electron microscope. An arylphorin dissociation intermediate identified as a trimer (1/2 x 6) was isolated; its possible quaternary structure is discussed on the basis of electron micrographs. The epitope of monoclonal antibody Ec-7 directed against tarantula (Eurypelma californicum) hemocyanin subunit d and also reactive to Calliphora arylphorin was traced to a highly conserved peptide of 27 amino acids localized in the center of the protein. The primary structure of Calliphora arylphorin as published in our preceding paper (Naumann and Scheller 1991) is compared in detail to the sequences of spider and spiny lobster hemocyanin. This revealed a basic framework of 103 strictly conserved amino acids. Isofunctional exchanges are proposed for another 76 positions. On the basis of these similarities, and the published three-dimensional model of spiny lobster hemocyanin, a detailed model of the quaternary structure of Calliphora arylphorin is presented. A second larval storage protein previously termed protein II was purified from Calliphora hemolymph. It was demonstrated to be a 500 kDa hexamer of 83 kDa subunits. In the electron microscope it shows a cubic view 9 nm in length with a large central hole and a rectangular view (9 x 10 nm) with a large central cavity. A morphologically very similar hemolymph protein was detected in Drosophila melanogaster larvae. From its structural appearance it is uncertain whether protein II belongs to the hemocyanin superfamily or not.

Amino Acid Sequence↗
PubMed1992

Electron microscopy of a double helical tubular filament in keyhole limpet (Megathura crenulata) hemolymph.

A approximately 25 nm hollow double helical filament has been detected ultrastructurally in the cell-free supernatant from hemolymph of the keyhole limpet Megathura crenulata (Gastropoda: Prosobranchia: Fissurellidae). Subsequently, much higher concentrations of this material were found in the cell pellet from hemolymph. Both negative staining and thin sectioning have been performed in an attempt to obtain a preliminary structural characterization of this new filament. It is proposed that the filaments are released or secreted from blood hemocytes in response to bleeding, but it has not been possible to define absolutely an intracellular organelle containing this material. It is shown that the hollow filaments, which have fine fibrillar surface extensions, are readily distinguishable from collagen fibrils and from tubular (multi-decameric) keyhole limpet hemocyanin (KLH).

Actin Cytoskeleton↗
PubMed1989

Allosteric oxygen-binding properties of reassembled tarantula (Eurypelma californicum) hemocyanin with incorporated apo- or met-subunits.

4x6-meric hemocyanin of the tarantula Eurypelma californicum was dissociated into subunits; one type of subunit was removed by immunoaffinity chromatography and replaced by its apo- or met-form. The mixture was reassembled and the reconstituted 4x6-mers were isolated. This was performed for subunits a, bc, d, e, f and g, respectively. It was verified by crossed immunoelectrophoresis that each type of subunit including the modified one, was incorporated in the reassembled 4x6-mers. Oxygen binding curves of the purified reconstituted 4x6-mers were recorded at different pH values (pH 7.0-9.0; 20 degrees C). Half-saturation pressures (P50) and the cooperativities were calculated using unmodified, reassembled 4x6-mers as reference. In all cases, incorporation of a met-subunit increased oxygen affinity. In contrast, incorporation of an apo-subunit either slightly decreased oxygen affinity (bc, f and d) or had no detected influence (others). The Bohr effect remained more or less unchanged in every case. Cooperativity was generally decreased. The met-modification of subunit d had the strongest effect. No significant differences could be observed between the respective met- and apomodification, except for experiments with subunit d. Generally, the value of hmax exceeded h50 by a factor of 1.3 to 1.5. pH-sensitivity of cooperativity was distinctly influenced depending on the modified subunit. The strongest effect was observed for subunit bc. Our results demonstrate, for the first time, that each subunit of tarantula hemocyanin is involved in the allosteric processes. Apparently, they uniformly contribute to oxygen affinity and Bohr effect, but distinctly to cooperativity and pH sensitivity of the latter.

Allosteric Regulation↗
PubMed1988

Hemocyanins in spiders. XXII. Range of allosteric interaction in a four-hexamer hemocyanin. Co-operativity and Bohr effect in dissociation intermediates.

The range of allosteric interaction in the 24-meric hemocyanin from the tarantula Eurypelma californicum was studied by measuring the oxygen-binding properties of defined oligomeric fragments. Dissociation intermediates comprising 19, 12, 7 or 6 subunits were obtained by incubation of native hemocyanin with 10 mM-cysteine at pH 4.4, with 40 mM-dithiothreitol at pH 7 or 8, by short-term alkaline (pH 9.6) treatment or by treatment with 4 M-urea. These could be stabilized by returning to neutral buffer conditions and, in the case of the dodecamer, by carboxymethylation. Conditions were chosen so that part of the starting material remained intact to serve as control in the oxygen-binding measurements. Oxygen equilibrium curves were obtained by a very sensitive fluorimetric/polarographic method. Oxygen affinity and the magnitude of the Bohr effect remain constant from the hexamer up to the control four-hexamer. Co-operativity, in contrast, increases with aggregate size in two steps: n (hexamer) = n (heptamer) less than n (dodecamer) = n (19-mer) less than n (4-hexamer). The characteristic pH-dependence of nH is absent in the hexa- and heptamer but is weakly indicated in the dodecamer, and fully established in the four-hexamer. Results from different preparations are highly consistent, if nH is expressed as a percentage of the control values. Full co-operativity is reached only in the four-hexamer, disproving the dodecameric half-molecule (the smallest repeating unit) as the allosteric unit. The stepwise increase in co-operativity appears to be correlated with higher levels of symmetry in the hierarchy of quaternary structure.

Animals↗
PubMed1988

Localization of cytokeratins in tissues of the rainbow trout: fundamental differences in expression pattern between fish and higher vertebrates.

Using a panel of antibodies against different cytokeratins in immunofluorescence microscopy on frozen tissue sections and two-dimensional gel electrophoresis of cytoskeletal proteins from these tissues, we have studied the tissue distribution of cytokeratins in a fish, the rainbow trout Salmo gairdneri. We have distinguished at least 14 different cytokeratin polypeptides in only a limited number of tissues, thus demonstrating the great complexity of the cytokeratin pattern in a fish species. The simplest cytokeratin pattern was that present in hepatocytes, comprising one type-II (L1) and two type-I (L2, L3) polypeptides that appear to be related to mammalian cytokeratins 8 and 18, respectively. Two or all three cytokeratins of this group were also identified in several other epithelial tissues, such as kidney. Epithelia associated with the digestive tract contained, in addition, other major tissue-specific cytokeratins, such as components D1-D3 (stomach, intestine and swim bladder) and B1 and B2 (biliary tract). With the exception of D1, all these polypeptides were also found in a cultured cell line (RTG-2). Epidermal keratinocytes contained D1 and six other major cytokeratins, termed E1-E6. The most complex cytokeratin pattern was that found in the gill epithelium. Surprisingly, antibodies specific for cytokeratins of the L1-L3 group also reacted with certain cell-sheet-forming tissues that are not considered typical epithelia and in higher vertebrates express primarily, if not exclusively, vimentin. Such tissues were (a) endothelia, including the pillar cells of the "gill filaments", (b) scale-associated cells, and (c) the ocular lens epithelium, and also several nonepithelial cell types, such as (d) fibroblasts and other mesenchymal cells, (e) chondrocytes, (f) certain vascular smooth muscle cells, and (g) astroglial cells of the optic nerve. The differences between the patterns of cytokeratin expression in this fish species and those of higher vertebrates are discussed. It is concluded that the diversity of cytokeratins has already been established in lower vertebrates such as fish, but that the tissue-expression pattern of certain cytokeratins has been restricted during vertebrate evolution. We discuss the value of antibodies specific for individual cytokeratin polypeptides as marker molecules indicating cell and tissue differentiation in fish histology, embryology, and pathology.

Animals↗
PubMed1985

Immunological correspondence between arthropod hemocyanin subunits. II. Xiphosuran (Limulus) and spider (Eurypelma, Cupiennius) hemocyanin.

The hemocyanins of the horseshoe crab Limulus polyphemus (48-mer), the tarantula Eurypelma californicum (24-mer), and the lycosid spider Cupiennius salei (dodecamer, hexamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Among the subunits a to g of Eurypelma on the one side, and I to VI of Limulus on the other, a number of cross-reactions were obtained which agree with the topologic subunit positions in the published models of quaternary structure: a = II, b-c = V-VI, d = IV, e = I, f = IIIb, g = IIIa (IIa). However, cross-reactivity was only strong in the following combinations: a/II, d/IV, b-c/V-VI (the monomers of the two heterodimers could not be correlated individually). A rather weak cross-reaction was obtained in the case of e/I and g/IIIa (IIa); a cross-reaction between f and IIIb was almost undetectable. On the other hand, f/IV clearly cross-reacted, and so did e/IIIa (IIa), which apparently is not in agreement with the two models of quaternary structure. These unexpected relationships, however, indicate the possible phylogeny of the subunits. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunit IV of Limulus and, moreover, revealed common antigen determinants present on these subunits. Denaturation of hemocyanin subunits of the three species with 8M urea yielded a completely different immunological behavior in that in all intra- and interspecific combinations the reaction of immunological identity was obtained. The published models of quaternary structure and a possible subunit phylogeny of cheliceratan hemocyanins is discussed in view of the present results and the results of the preceding paper. [Markl, J. et al. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 619-631.]

Animals↗