Immunological Studies of Tissue Proteinases

Poole, A R

doi:10.1007/978-1-4615-7951-9_7

Cited by 4 publications

(1 citation statement)

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“…Total concentration and content of both inactive and active cathepsin D are determined using specific antibodies by the immunoenzymatic method ELISA [151,152] and by radioimmunoenzymatic methods [153,154] (Table 11).…”

Section: Determination Of Concentrationmentioning

confidence: 99%

Quantitative determination and localization of cathepsin D and its inhibitors.

Minarowska

Karwowska²,

Gacko³

2009

Folia Histochem Cytobiol.

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Cathepsin D (EC 3.4.23.5) is a lysosomal aspartyl endopeptidase, localized in all cells and tissues, except for mature erythrocytes [1][2][3]. Methods used to determine the activity, concentration and cellular distribution of cathepsin D, but not its inhibitors, have previously been the subject of literature reports [4][5][6][7][8][9][10][11][12]. However, since the time of their publication a number of new substrates and analytical techniques have been implemented. Structure, specificity, mechanism of actionCathepsin D is synthesized in the rough endoplasmic reticulum as preprocathepsin D, built up of 412 amino acid residues [13][14][15]. As a result of cleavage of the 20-amino acid signal prepeptide, it is converted into procathepsin D which undergoes glycosylation and disulphide bridges are formed in its molecule. Procathepsin D is transported from cisterns of the rough endoplasmic reticulum to the Golgi apparatus, from which, with the involvement of mannoso-6-phosphate (M-P-6) receptors, it is transferred to primary lysosomes [16,17]. As the M-6-P receptors are known to occur in the primary lysosomes but not in the mature ones, they can be used to distinguish between these two types of lysosomes [18]. In the acidic environment of the lysosomes (pH 4.5-5.5), due to autocatalytic cleavage of the 44-amino acid propeptide from the N-terminal molecule, procathepsin D is converted into the active one-chain form. The actions of cysteine proteinase, aminopeptidases and carboxypeptidases lead to the formation of an active two-chain form of cathepsin D (Fig. 1). These chains are bound by hydrophobic bonds. The molecular weight of the ultimate mature form of cathepsin D is 48 (14+34) kDa. The proteolytic activities of the one-chain and two-chain forms are very similar [19,20]. Modification of the polypeptide chain, different oligosaccharide composition types and phosphorylation/dephosphorylation in the amino saccharide residues contribute to marked molecular heterogenicity of cathepsin D and cause differences in isoelectric points of the respective isoenzymes between pH 4.5 -6.5 [21,22].The use of peptides with the known primary structure allows identification of amino acid residues that form peptide bonds cleaved by cathepsin D. For this purpose, synthetic peptides [23] and chains A and B of bovine insulin can be used (Fig. 2). Cathepsin D cleaves the peptide bonds found within the polypeptide chain, formed by carboxyl groups of the hydrophobic amino acid residues: aromatic -trypto-

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Section: Determination Of Concentrationmentioning

confidence: 99%