Bernd Wiederanders

Antigenic expression of cathepsin B in aged human brain

The lysosomal thiol proteinase, cathepsin B, has been localized in different regions of aged human brain by use of the peroxidase-antiperoxidase technique. Cathepsin B-immunoreactive material was detected in multiple neurons of human hippocampus, neocortical area A 10, prefrontal gyrus and nuc. basalis of Meynert as well as in single white matter astrocytes. In brains of Alzheimer disease-affected subjects cathepsin B was revealed in neuritic plaques too. Possible functional consequences with regard to normal aging, neuropeptide metabolism and pathological changes are discussed.

Accumulation of inactive cathepsin D in old rats

From the 2nd to the 29th month of life seven different rat organs (liver, heart, kidney, lung, spleen, skeletal muscle, intestinal mucosa) show a continuous increase in the concentration of protein reacting with rabbit antiserum against rat liver cathepsin D. However, the activity of aspartate proteinases, probably mainly cathepsin D, does not show the same increase. This result is interpreted as accumulation of enzymatically inactive but immunologically reactive cathepsin D. This phenomenon, which is well known for some cytosolic enzymes in senescent animals, is first reported to occur for a lysosomal proteinase as well.

The processing of a cathepsin L precursor in vitro.

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.

Primary structure of bovine cathepsin S. Comparison to cathepsins L, H, B and papain.

The primary structure of bovine cathepsin S was determined by combining results of protein and peptide sequencing with the sequence deduced from nucleic acid sequencing. Using polymerase chain reaction (PCR) technology, cDNA clones commencing at amino acid 22 of the mature enzyme and continuing through the 3′ untranslated region of bovine cathepsin S mRNA were isolated and sequenced. The open reading frame in these overlapping clones correctly predicts the determined amino acid sequence of 13 tryptic peptides derived from purified bovine spleen cathepsin S. The deduced amino acid sequence shows that mature bovine cathepsin S consists of 217 amino acids corresponding to a molecular weight of 23.7 kDa. Cathepsin S belongs to the papain superfamily of lysosomal cysteine proteinases and shares 41% identity with papain. Amino acid sequence identities of bovine cathepsin S to human cathepsins L, H, and B are 56%, 47% and 31% respectively.

Cell type-specific distribution of cathepsin B and D immunoreactivity within the rabbit retina

The cellular localization of cathepsin B and D immunoreactivity was demonstrated at the light microscopic level in the retina of adult rabbits by use of the peroxidase-antiperoxidase technique. Antisera were raised against rat liver enzymes. Whereas cathepsin D immunoreactivity was confined to Müller (glial) cells, cathepsin B was demonstrated in some, but not all, neuronal cell types. It is proposed that the two enzymes might carry different functions within the neuronal versus glial compartment.

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

SummaryThe 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.

Distribution of cathepsin D immunoreactivity in the central nervous system of rat and selected brain regions of man

The regional distribution and cellular localization of cathepsin D immunoreactivity was demonstrated at the light microscopic level in the CNS of rat and man by use of unlabelled immunoenzyme technique. A wide but uneven distribution was substantiated for the rat brain. Furthermore, we present evidence that antiserum produced against rat liver enzyme is capable of recognizing cathepsin D in human brain.

Cathepsin D, B, and H in rat brain as demonstrated by immunohistochemistry.

Monospecific antisera against cathepsins B, D, and H were used to immunolocalize these proteinases in neural structures of rat brain. Cathepsins B and D were found to be largely co-localized in nerve cells. Cathepsin H could not be identified by use of immunocytochemistry.

PROTEIN CATABOLISM IN RAT LIVER CELLS

Publisher Summary This chapter discusses protein catabolism in rat liver cells. The use of in vivo short- and long-lived proteins as substrates in vitro allows proving whether the conditions chosen in vitro might be a sufficient simulation of conditions in living cells, and also whether the proteinase used is important for the selective breakdown of short-lived substrate proteins. The protein degradation occuring in vitro in the microsomal fraction must not sufficiently simulate the in vivo process, because a selective autolytic and lysosomal degradation of the in vivo long-lived membrane proteins. Investigations of the primary steps of intracellular protein catabolism must include all organelles, especially the organelles in the microsomal fraction, the energy requirement, the possible role of microtubules and the distinction between basal and enhanced intracellular proteolysis.

Fraktionierung hydroxyprolin-haltiger harnpeptide mittels sephadex-gelfiltration

Abstract Fractionation of Hydroxyproline-containing Urinary Peptides by Sephadex Gel Filtration Concentrated urine samples from 11 healthy persons and from 29 persons with disorders of connective tissue, skin or bone, as well as urine samples from 10 rats, 5 of them lathyritic, were examined. The samples were fractionated by means of gel filtration on Sephadex G-25- The peptide-bound hydroxyproline was determined in the fractions. 25% of hydroxyproline in man and 20% of that in rats was excreted as part of long-chain peptides. We could find no significant variation from this relation in disorders with alteration of the collagen metabolism. We estimated the molecular weights of the long-chain peptides at 1000–6500. The significance of these findings is discussed with reference to the degradation of collagen.