Human tumour cathepsin B. Comparison with normal liver cathepsin B

Abstract

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.

Cathepsin B Is Inhibited in Mutant Cells Selected during Persistent Reovirus Infection

“…In addition to the absence of single-chain forms of cathepsin B in LX cells, it is also possible that these cells express an activity that inhibits double-chain forms of the enzyme, which seems likely because the double-chain form of cathepsin B is known to be enzymatically active (34,35,37,38,54). In a previous study, the single-chain and double-chain forms of cathepsin B were separated by ion exchange chromatography and tested for catalytic activity.…”

“…To determine the underlying mechanism of the block to viral disassembly in LX cells, we studied the expression and activity of cysteine endocytic proteases cathepsin B, cathepsin H, and cathepsin L. Both the mature singlechain and double-chain forms of these cathepsins are enzymatically active (34,35,37,38). L cells expressed both single-chain and double-chain forms of each enzyme.…”

“…The heavy and light chains are linked by a disulfide bond, forming the double-chain form of the enzyme. Both the single-chain and double-chain forms of cathepsin B and cathepsin L are enzymatically active, whereas the proenzyme forms of these enzymes are not (34,35,37,38).…”

Functional expression of recombinant human stefin A in mammalian and bacterial cells

“…Membranes containing the electrophoretically transferred proteins were developed with an enhanced chemiluminescence western blot detection system using 10% nonfat dry milk and 0.05% Tween-20 as blocking agents, rabbit IgG raised against rat cystatin α or polyclonal antibody to human cystatin C, as primary antibodies, and horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody according to our published protocols (30).…”

Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH

“…The catB selective inhibitor, CA074 was purchased from Peptides International, Inc. (Louisville, KY, USA). Rabbit anti-human liver catB IgG was prepared as previously described (Moin et al, 1992). Myosin heavy-chain antibody (MF20) was generously provided by Ilona Skerjanc (University of Western Ontario, London, ON, Canada).…”