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P Bohley

Publications and source records attributed to P Bohley.

At least 19 recordsLinked to original sources
PubMed2001

Proteome analysis by three-dimensional protein separation: turnover of cytosolic proteins in hepatocytes.

We performed a three-dimensional separation of pulse-chase dual-labelled rat liver cytosolic proteins using hydrophobic interaction chromatography, isoelectric focusing, and SDS gel electrophoresis. Due to very different expression rates but similar size and pI of rat liver cytosolic proteins, we demonstrate the impossibility of successful two-dimensional separations of such complex protein mixtures. A pre-fractionation of proteins by hydrophobic interaction chromatography is therefore recommended prior to two-dimensional gel electrophoresis. Our studies confirmed the correlation between protein turnover rates and surface hydrophobicity.

Animals↗
PubMed2000

Highly potent inhibitors of human cathepsin L identified by screening combinatorial pentapeptide amide collections.

By screening a combinatorial pentapeptide amide collection in an inhibition assay, we systematically evaluated the potential of 19 proteinogenic amino acids and seven nonproteinogenic amino acids to serve as building blocks for inhibitors of human cathepsin L. Particularly efficient were aromatic, bulky, hydrophobic amino-acid residues, especially leucine, and positively charged residues, especially arginine. Building blocks for potential inhibitory peptides were combined by random selection from their activity pattern. This random approach for the design of inhibitors was introduced to compensate for the inaccuracy induced by shifted docking of combinatorial compound collections at the active center of cathepsin L. Thereby, we obtained structurally defined pentapeptide amides which inhibited human cathepsin L at nanomolar concentrations. Among the most potent novel inhibitors, one peptide, RKLLW-NH2, shares the amphiphilic character of the nonamer fragment VMNGLQNRK of the autoinhibitory, substrate-like, but reverse-binding prosegment of human cathepsin L which blocks the active center of the enzyme. Obviously, RKLLW-NH2 carries the functions that are important for enzyme-peptide interaction in a condensed form. This hypothesis was confirmed by structure-activity studies using truncated and modified pentapeptides.

Amides↗
PubMed1995

The fates of proteins in cells.

Nascent polypeptide chains are in a dangerous situation as soon as they leave their place of birth, the channel of the large ribosomal subunit: more than 20 different pathways for the degradation of proteins exist in cells. Chaperones protect and guide the young protein molecules and support their correct foldings. Targeting signals direct the proteins to the organelles of their destination. The lysosome is the site of random degradation, while the proteasome is highly selective. Although these two organelles provide the most important pathways for the degradation of long- and short-lived proteins, other pathways with roles in deciding the fate of cellular proteins must also be considered.

Amino Acid Sequence↗
PubMed1993

Preparation and characterization of monoclonal antibodies against ornithine decarboxylase.

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC (Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345-360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355-360) portion whereas MP16-3 reacted with the ODC-(345-350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

Amino Acid Sequence↗
PubMed1992

Proteases and proteolysis in the lysosome.

Proteins sequestered by a non-selective bulk process within the lysosomes turn over with an apparent half-life of about 8 minutes and this rapid lysosomal proteolysis is initiated by endopeptidases, in particular by the cathepsins D and L. We describe also the cathepsins B and H which show mainly exopeptidase and only low endopeptidase activity. Especially cathepsin H is most probably the only lysosomal aminopeptidase in many cell types. Additionally, the properties of other mammalian lysosomal endo- and exopeptidases are compared. Finally, we discuss some of the conditions for the action of lysosomal proteases as the low intralysosomal pH, the high part of lysosomal thiol groups and the absence of intralysosomal proteinase inhibitors.

Animals↗
PubMed1992

Autophagy and other vacuolar protein degradation mechanisms.

Autophagic degradation of cytoplasm (including protein, RNA etc.) is a non-selective bulk process, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lives. The initial autophagic sequestration step, performed by a poorly-characterized organelle called a phagophore, is subject to feedback inhibition by purines and amino acids, the effect of the latter being potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, on intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy to a variable extent, and that the amphisome may play a central role as a collecting station for material destined for lysosomal degradation. Protein degradation can also take place in a 'salvage compartment' closely associated with the endoplasmic reticulum (ER). In this compartment unassembled protein chains are degraded by uncharacterized proteinases, while resident proteins return to the ER and assembled secretory and membrane proteins proceed through the Golgi apparatus. In the trans-Golgi network some proteins are proteolytically processed by Ca(2+)-dependent proteinases; furthermore, this compartment sorts proteins to lysosomes, various membrane domains, endosomes or secretory vesicles/granules. Processing of both endogenous and exogenous proteins can occur in endosomes, which may play a particularly important role in antigen processing and presentation. Proteins in endosomes or secretory compartments can either be exocytosed, or channeled to lysosomes for degradation. The switch mechanisms which decide between these options are subject to bioregulation by external agents (hormones and growth factors), and may play an important role in the control of protein uptake and secretion.

Animals↗
PubMed1991

Post-translational arginylation and intracellular proteolysis.

Cellular proteins may be designated to fast degradation by their N-terminal amino acids, and especially a N-terminal arginine residue should have an extremely destabilizing effect on cytosol proteins. We investigated the post-translational arginylation of cytosol proteins and especially of ornithine decarboxylase (ODC) by the cytosolic enzyme arginyl transferase by incubation with radioactive L-arginyl-tRNA and isolation of ODC with our monoclonal antibody. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic proteins and Edman-degradation of this ODC showed that the post-translational arginylation occurred only at the L-amino-end of the enzyme. The inhibitor of arginyltransferase, L-Glutamyl-L-Valyl-L-Phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. This post-translational arginylation of ODC and also of other cytosol proteins is reversible. At least 25 different cytosol proteins in addition to ODC can be arginylated in hepatocytes, and at least 15 different proteins can be arginylated in Dictyostelium discoideum. The arginylated proteins are much more rapidly degraded by cellular proteinases, especially by calpains, than those cytosolic proteins which are not arginylated.

Acyltransferases↗
PubMed1990

Post-translational arginylation of ornithine decarboxylase from rat hepatocytes.

Ornithine decarboxylase (ODC) was purified 6500-fold from NMRI mouse kidneys under conditions designed to inhibit degradation by proteinases. The enzyme was homogeneous by SDS/polyacrylamide-gel electrophoresis, and the specific activity was among the highest reported. The yield was 70%. A monoclonal antibody against this preparation was generated and used in studies to investigate the half-life of ODC in cultured rat hepatocytes labelled with [35S]methionine. This value was 39 +/- 4 min and was unchanged when either NH4Cl (as a lysosomotropic agent) or leupeptin (as a lysosomal proteinase inhibitor) was added to the culture medium. Thus the intracellular turnover of ODC in cultured hepatocytes occurs mainly in extra-lysosomal compartments. Arginylation of rat ODC was investigated in vitro by incubation with L-[3H]arginyl-tRNA, and the incorporation of the label was compared with that of total cytosolic proteins. Arginylated ODC had a specific radioactivity 8600 times that of the bulk of cytosolic protein. Edman degradation of this ODC showed that the post-translational arginylation occurred only at the alpha-amino end of the enzyme. The inhibitor of arginyl-tRNA:protein arginyltransferase (EC 2.3.2.8), L-glutamyl-L-valyl-L-phenylalanine, increased the half-life of ODC in cultured hepatocytes from 39 min to more than 90 min. The possible significance of the preferential post-translational arginylation of ornithine decarboxylase to its rapid turnover is discussed.

Animals↗
PubMed1990

Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes.

Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.

Animals↗
PubMed1990

Very fast purification of ornithine decarboxylase with high yield from mouse kidney and generation of a monoclonal antibody.

Based on methods for ornithine-decarboxylase purification published previously we developed an improved procedure for purification of the enzyme from the kidneys of testosterone-treated NMRI mice. Advantages of the new procedure are, that inactivation of the enzyme during purification is largely reduced by fast methods for purification and by the use of proteinase inhibitors. That way we got pure ornithine decarboxylase within 60 h with a yield of about 70%. A part of the highly purified ornithine decarboxylase was used for the generation of monoclonal antibodies.

Animals↗
PubMed1988

Surface hydrophobicity, arginylation and degradation of cytosol proteins from rat hepatocytes.

In vitro 14C-prelabelled cytosol proteins from rat hepatocytes were incubated with [3H]arginyl-tRNA in ATP-Tris-Mg-KCl-dithioerithrol medium and arginyltransferase, subsequently treated with RNase A, and the double-labelled proteins were isolated by gel filtration. The affinity of these [3H]arginylated-14C-labelled cytosol proteins to hydrophobic surfaces was investigated with octyl-Sepharose, phenyl-Sepharose and with FPLC on phenyl-Superose (HR 5/5). All 3H/14C-ratios of the proteins in the column fractions show that arginylated proteins bind preferentially to the hydrophobic matrices: the fractions eluted first show low 3H/14C-ratios, and after addition of ethylene glycol and especially of Tween 80 the 3H/14C-ratios markedly increase. Furthermore, these arginylated proteins aggregate preferentially after incubation of the cytosol proteins for 2 h at 37 degrees C and are more rapidly degraded by endopeptidases.

Animals↗
PubMed1985

Localization of cathepsin H and its inhibitor in the skin and other stratified epithelia.

The rat-skin-derived cysteine proteinase, so-called BANA-hydrolase, which is capable of hydrolysing benzoylarginine naphthylamide and leucine naphthylamide was shown to be immunologically identical to cathepsin H purified from rat liver. The enzyme was immunocytochemically localized in the basal cell layer of rat epidermis. A natural inhibitor of cathepsin H with a molecular weight of about 13,000 was mainly localized in the keratinizing cell layers and showed only a weak reaction in the basal cells. Thus, cathepsin H appears to be a characteristic feature of the proliferating cell layer, whereas the cysteine-proteinase inhibitor is a characteristic feature of keratinizing cells.

Aminopeptidases↗
PubMed1984

The insulin and glucagon degrading proteinase of rat liver. Separation of the proteinase from the thiol-proteindisulfide oxidoreductases.

Insulin degrading enzymes of rat liver cytosol, the so-called insulin and glucagon degrading proteinase (IGP, EC 3.4.23.5), and two forms of the insulin degrading thiol-protein-disulfide oxidoreductase/isomerase (glutathione-insulin transhydrogenase, TPO, EC 1.8.4.2/5.3.4.1) were separated from each other and partially purified on DEAE-Sephadex. The highly purified proteinase was obtained by polyacrylamide gel electrophoresis of the DEAE-Sephadex-purified enzyme fraction and was used to produce monospecific antibodies to the IGP in rabbits. Strong evidence is given that the insulin and glucagon degrading proteinase is an autonomous enzyme existing in addition to the TPO forms in the cytosol of the liver. Combined action of the proteinase and the TPO system on radioiodinated insulin under various conditions in vitro revealed an independent and non-sequential degradation of insulin by these two enzyme systems.

Animals↗
PubMed1984

The insulin and glucagon degrading proteinase of rat liver: a metal-dependent enzyme.

Insulin and glucagon degrading proteinase (EC 3.4.23.5) purified from rat liver cytosol was characterized using radioiodinated insulin and glucagon as substrates. Maximum activity for breakdown of both hormones was found at pH 8.1. Thiol blocking reagents as well as indole derivatives inhibit the proteinase, whereas pepstatin, leupeptin, bestatin, elastatinal, antipain, chymostatin and phosphoramidon do not have any effect. Although the Km values and maximal velocities of insulin and glucagon breakdown deviate strongly from each other, the specificity constants (kcat/Km) for both substrates are nearly identical. The insulin and glucagon degrading proteinase, known as a thiol-dependent enzyme, was found to be also a metallo enzyme. Chelating agents, such as EDTA, EGTA, bipyridine and o-phenanthroline show a concentration dependent inhibition. The strongest inhibitor found was o-phenanthroline. Zn++, Co++, Mn++, and to a smaller extent Cd++ and Fe++, are capable of preventing the o-phenanthroline mediated inhibition. Removal of the protein-bound metal(s) results in a nearly total and irreversible loss of enzymatic activity.

Animals↗
PubMed1982

The ribosomal serine proteinase, cathepsin R. Occurrence in rat-liver ribosomes in a cryptic form.

Ribosomes have been shown to contain a proteolytic activity, characterized as an endopeptidase with serine in the active center. The enzyme has been given the name cathepsin R, following the recommendations of Barrett et al. (in a publication from the Cold Spring Harbor Laboratory, New York) for naming new proteinases. The present paper contains evidence that cathepsin R in rat liver ribosomes is present in a cryptic form. Upon dissociation of ribosomes to subunits (and to minor extent also by 0.5 M KC1 washes), the cryptic proteinase is released. Activation of the released cathepsin R is effected by equilibration with 2 M NaC1/0.05 M sodium acetate, pH 4.8. The molecular weight of free cathepsin R is 25 000-30 000.

Animals↗