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Biomedical subjects

L Kuehn

Publications and source records attributed to L Kuehn.

At least 37 records · Page 2Linked to original sources
PubMed1992

Biochemical properties of the proteasome from Thermoplasma acidophilum.

We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90 degrees C. The enzyme activity is enhanced severalfold by Mg2+ and Ca2+ at 25-500 mM. This increase in activity results primarily from a change in Km. The serine-proteinase inhibitors diisopropylfluorophosphate and 3,4-dichloroisocoumarin irreversibly inhibit the enzyme, obviously by modification of both the alpha and beta subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz-Leu-Leu-CH2Cl and Cbz-Ala-Ala-Phe-CH2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.

Amino Acid Sequence↗
PubMed1992

Evidence indicating that the multicatalytic proteinase of rabbit reticulocytes is not incorporated as a core enzyme into a 26 S proteinase complex.

We have reinvestigated the recent proposal that the multicatalytic proteinase, together with other components of reticulocyte lysate, may become incorporated into a very large, "26 S" proteinase complex via an ATP-dependent process. Different from these published results, we consistently isolate the multicatalytic proteinase as a 650,000 Da "20 S" multisubunit proteinase. Analysis on nondenaturing polyacrylamide gels of reticulocyte fractions containing the putative complexed form of the multicatalytic proteinase reveal that activity against succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin is associated with two groups of protein of different molecular mass. One migrates like multicatalytic proteinase purified to homogeneity, displays, on sodium dodecyl sulfate gels, a set of protein species in the range of 23,000-32,000 Da, characteristic of the multicatalytic proteinase, and is recognized by a monospecific antibody to the enzyme. In contrast, the activity associated with the higher molecular mass (26 S) proteinase complex lacks the typical multicatalytic proteinase subunits and is devoid of antigenic material, when tested with the antibody. These results confirm and extend our recent findings in mouse liver by showing that the multicatalytic proteinase is not a constituent of a 26 S proteinase complex.

Animals↗
PubMed1991

Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the '26 S' multienzyme complex.

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a '26 S' complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass '26 S' multienzyme complex. In all instances proteasomes are identified in their 'free' 650 kDa '20 S' form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described '26 S' proteinase. This '26 S' proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the '26 S' proteinase complex.

Amino Acid Sequence↗
PubMed1991

The multicatalytic proteinase (prosome, proteasome): comparison of the eukaryotic and archaebacterial enzyme.

Proteasomes isolated and purified from rat muscle tissue and from the archaebacterium Thermoplasma acidophilum have a very similar size and shape, but the subunit composition is less complex in the archaebacterium as compared to the eukaryotic particle. The archaebacterial enzyme contains a catalytic site with chymotryptic specificity, which is inhibited by serine proteinase inhibitors and clearly differs from the eukaryotic particle which has a minimum of three catalytic sites for peptide bond hydrolysis of a yet undefined mechanism.

Amino Acid Sequence↗
PubMed1991

Tissue-specific changes of multicatalytic proteinase activity in the fasted rat.

During a three-day fast, followed by four days of refeeding, the content of the multicatalytic proteinase as well as hydrolyzing activity towards Suc-Leu-Leu-Val-Tyr-7-amino-4-methylocoumarin (SLLVT-MCA) was measured in various rat tissues. When compared with normal rats, the MCP content, as determined by immunochemical techniques, was unchanged over the entire experimental period in the three tissues examined: gastrocnemius muscle, thymus and testis. By contrast, a differential response was observed in the three tissues with respect to specific and total SLLVT-MCA splitting activity: for thymus and testis, these values were again unchanged, whereas in gastrocnemius muscle, both specific and total enzyme activity fell by almost 70% on day three of fasting but returned to control values on day four of refeeding. This change in activity was not due to the accumulation or degradation of a specific proteinase inhibitor. Data demonstrate that, in association with the insulin-deficient state of starvation, the activity of the multicatalytic proteinase shows an adaptive behaviour which becomes manifest in some but not in other tissues.

Animals↗
PubMed1990

Effect of the multicatalytic proteinase (prosome) on translational activity in rabbit reticulocyte lysates.

In a message-dependent reticulocyte lysate translation system, incorporation of [3H]leucine into acid-insoluble protein is increased following selective removal of the multicatalytic proteinase (MCP) with a monospecific antibody. Re-addition of active proteinase to previously depleted lysates reverses this effect in that the same low levels of translational product are measured as in untreated lysates. Addition of histone-stimulated MCP further depresses the level of protein product. Conversely, lysates supplemented with inactivated MCP retain the higher level of translational activity which is measured after precipitation of the enzyme with antibody. In these lysates, the effect of the antibody on translational activity is inversely correlated with that on hydrolytic activity towards [14C]methylcasein or N-succinyl-Leu-Leu-Val-Tyr-4-methyl-7-coumarylamide, two substrates of the MCP. These results showing that the MCP is capable of modulating translational activity in vitro, suggest an important role of this molecule in the in vivo translational process.

Amino Acid Sequence↗
PubMed1989

The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria.

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.

Animals↗
PubMed1989

ATP-activated, high-molecular-mass proteinase-I from rat skeletal muscle is a cysteine proteinase-alpha 1-macroglobulin complex.

From rat skeletal muscle tissue we have isolated and purified a proteolytic activity of molecular mass 750 kDa. The enzyme, designated 'proteinase I', which has been found to be located in capillaries of skeletal muscle tissue, catalyzes the hydrolysis of Z-Phe-Arg-MCA and [14C]methylcasein and this process is activated about 2-fold by ATP. As judged by SDS-polyacrylamide gel electrophoresis the subunit pattern of 'proteinase I' is similar to alpha-macroglobulin. Immunoelectrophoretic analyses of 'proteinase I' with antisera to rat alpha 1-macroglobulin, alpha 2-macroglobulin, and rat liver cathepsins reveal that this high-molecular-mass proteinase is a complex of alpha 1-macroglobulin and the cysteine proteinases cathepsin B, H and L. A similar 'proteinase' has been isolated from rat serum. Two ATP-activated high molecular-mass proteinases that have been previously identified in liver and heart muscle by other investigators equally show a positive immunological reaction with the antiserum raised against 'proteinase I'. From these data, together with results presented in an accompanying paper (Kuehn, L., Dahlmann, B., Gauthier, F. and Neubauer, H.-P. (1989) Biochim. Biophys. Acta 991, 263), we conclude that the ATP-stimulated high-molecular-mass proteolytic activity is partly due to the presence of a complex of alpha-macroglobulin and cysteine proteinases.

Adenosine Triphosphate↗
PubMed1989

High-molecular-mass proteinases in rabbit reticulocytes: the multicatalytic proteinase is an ATP-independent enzyme and ATP-activated proteolysis is in part associated with a cysteine proteinase complexed to alpha 1-macroglobulin.

We have investigated the proteolytic degradation of [14C]methylcasein and 125I-labeled bovine serum albumin at pH 7.8 and 37 degrees C by lysates of rabbit reticulocytes purified from rabbit blood by two different procedures. (I) Lysates obtained from reticulocytes after removal of plasma and buffy coat as well as after washing of cells, degraded casein and albumin, and released from the two substrates 1.3%/h and 0.4%/h, respectively, of acid-soluble radioactivity. The activity towards both substrates was stimulated about 4-fold by ATP/Mg2+. Chromatography of whole blood on a column of cellulose prior to washing and lysis of cells had profound but differential effects on these activities in that stimulation of casein-degradation by ATP/Mg2+ was almost completely lost, whereas degradation of albumin, albeit at a low rate, was measurable in the presence of ATP/Mg2+ only. (II) Degradation of casein by these lysates is largely inhibited by a monospecific antibody against rabbit multicatalytic proteinase, whereas digestion of albumin is not affected by the antibody, either in the presence or absence of ATP/Mg2+. The latter activity is partially inhibited by a specific antibody against rabbit alpha 1-macroglobulin. (III) The immunoreactive amount of multicatalytic proteinase is about 1.2 micrograms per mg of lysate protein and almost identical in the two lysates. In contrast, the immunologically detectable levels of alpha 1-macroglobulin vary and are much lower in reticulocyte-lysates following chromatography on cellulose than in lysates from washed reticulocytes. (IV) Caseinolytic activity of multicatalytic proteinase, purified from rabbit reticulocyte lysate, is not activated by ATP/Mg2+ and the enzyme is proteolytically inactive towards albumin. On the other hand, a complex consisting of the proteinase inhibitor alpha 1-macroglobulin and the cysteine proteinase, cathepsin B, does degrade both substrates at pH 7.8, in an ATP/Mg2+-activated fashion. From these results it is concluded that the multicatalytic proteinase is an ATP-independent enzyme and a cellular constituent of rabbit reticulocytes whereas the activity stimulated by ATP/Mg2+ appears to be associated, at least in part, with a cysteine proteinase complexed to alpha 1-macroglobulin.

Adenosine Triphosphate↗
PubMed1989

Gender differences in schizophrenia: rehospitalization and community survival.

This study tested the hypothesis that schizophrenic men experience a poorer course than schizophrenic women. A representative sample from Hannover, FRG, including 278 first admission DSM-III schizophrenics, were followed for three years. Findings demonstrated that schizophrenic women experience fewer rehospitalizations, shorter lengths of stay, and survive longer in the community than schizophrenic men. Implications of the role of gender for schizophrenia are discussed.

Adult↗
PubMed1988

Electron microscopy and image analysis of the multicatalytic proteinase.

One electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end-on (ring shaped) or side-on (rectangular shaped). For aurothioglucose, ammonium molybdate- and phosphotungstate-stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate-staining reveals ring-shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side-on views of the particles reveals a tripartite, reel-shaped structure. Within the ring-like, end-on projections of ammonium molybdate-stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six-fold symmetry.

Animals↗
PubMed1988

Drosophila small cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase.

All eukaryotic cells so far analysed contain 19S particles which share a cylinder-like shape and are composed of a set of proteins of relative molecular mass ranging typically from 19,000 to 36,000 (refs 1-10). Proposed functions have included synthetase activity, transfer RNA processing or messenger RNA repression, but their biological importance remains obscure. A multicatalytic proteinase (MCP) of similar size and shape has been isolated from mammalian tissues. The apparent similarities of these high molecular weight complexes suggest a biochemical and functional homology between the small cytoplasmic 19S particle from Drosophila melanogaster (19S-scRNP) (ref. 7) and rat MCP (ref. 14). By means of electron microscopy, immunological techniques, RNA identification and proteinase activity assays, we were able to show that the two structurally similar complexes are immunologically related ribonucleoproteins (RNPs) with similar proteolytic activity.

Animals↗
PubMed1988

Changes in proteinase/proteinase inhibitor levels in rat skeletal muscle tissue during diabetes and fasting.

The skeletal muscle content of three rat proteinase inhibitors, a 1-proteinase inhibitor, contrapsin and a 1-cysteine proteinase inhibitor was measured by immunochemical techniques following streptozotocin-induced diabetes. When compared with normal rats, a 1-cysteine proteinase inhibitor and a 1-proteinase inhibitor levels remained essentially unchanged, whereas the content of rat contrapsin was reduced by nearly 80% after the onset of diabetes. Similarly, fasting of rats for three days resulted in a lowering of the levels of contrapsin in skeletal muscles. Under these conditions, levels of chymotrypsin-like activity (chymase) were increased by 150%, whereas the content of the trypsin-like, neutral proteinase was unchanged. Kinetic studies in vitro with Tosyl-Gly-Pro-Arg-4-nitroanilide as substrate showed no inhibition of the trypsin-like proteinase by a 1-proteinase inhibitor, while contrapsin inhibited the enzyme with a Ki value of 40nM. The changing pattern of these proteinases and their potential inhibitors (chymase/a 1-proteinase inhibitor and trypsin-like proteinase/contrapsin) may be a factor contributing to muscle wasting as observed in diabetes and fasting.

Animals↗
PubMed1986

Size and shape of the multicatalytic proteinase from rat skeletal muscle.

The multicatalytic proteinase from rat skeletal muscle, a non-lysosomal high molecular weight enzyme active at neutral to alkaline pH, has been examined in the electron microscope as well as by dynamic laser light scattering. Both methods reveal monodisperse particles. Electron micrographs show a cylinder-shaped complex with a diameter of 11 nm and a length of 16 nm in negatively stained, and a diameter of 9.6 nm and a length of 14.3 nm in freeze-dried, heavy metal replicated specimens. The molecule is composed of four rings or disks.

Animals↗
PubMed1986

Studies on the multicatalytic proteinase from rat skeletal muscle.

The multicatalytic proteinase purified from rat skeletal muscle was shown by electron microscopy to be a uniform cylinder-shaped protein particle. As the enzyme activities are enhanced by free fatty acids in vitro, we tested, by in vivo perfusion, whether high concentrations of free fatty acids increased the multicatalytic proteinase activities in skeletal muscle tissue. Perfusion of rat hindquarters with plasma containing 1.5 mM oleic acid bound to albumin led to a 30-40% increase of the proteinase activities in gastrocnemius muscle. This increase was statistically not significant when compared to control rats perfused with plasma containing fatty acid free albumin. These results are discussed under the following aspects: 1. the in vivo concentration of non-esterified fatty acids within the muscle cell is not known; 2. the uptake of free fatty acids by the muscle cell is too low, or the metabolism of free fatty acids taken up by the cell is too fast to allow activation of the multicatalytic proteinase; 3. the free fatty acids are bound to other proteins (e.g. fatty acid binding protein), which abolish their ability to activate the proteinase.

Animals↗