Abstract
Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatography on insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. Physicochemical and immunochemical measurements identified one as cystatin A, one as cystatin B, one as cystatin C, one as cystatin S, and one as low molecular weight kininogen. The sixth inhibitor displayed immunochemical cross-reactivity with salivary cystatin S but had a different pI (6.85 versus 4.68) and a different (blocked) N-terminal amino acid. This inhibitor was tentatively designated cystatin SU. The isolated inhibitors accounted for nearly all of the cysteine proteinase inhibitory activity of the urinary pool used as starting material. The enzyme inhibitory properties of the inhibitors were investigated by measuring inhibition and rate constants for their interactions with papain and human cathepsin B. Antisera raised against the inhibitors were used in immunochemical determinations of their concentrations in several biological fluids. The combined enzyme kinetic and concentration data showed that several of the inhibitors have the capacity to play physiologically important roles as cysteine proteinase inhibitors in many biological fluids. Cystatin C had the highest molar concentration of the inhibitors in seminal plasma, cerebrospinal fluid, and milk; cystatin S in saliva and tears; and kininogen in blood plasma, synovial fluid, and amniotic fluid.
Highlights
Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatographyon insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption
All human biological fluids investigated show cysteine proteinase-inhibiting activity, but it is not known whether the above-mentioned inhibitors accounftor the major part of this activity; neither is itknown whether theirrelative concentrations vary between different biological fluids and if they have the capacity toplay important physiological roles as cysteine proteinase inhibitors
Isolation and Characterization of Urinary Cysteine Proteinase Inhibitors-Urine from one single individual with mixed glomerular-tubular proteinuria was used in the fractionation procedure described below
Summary
Six cysteine proteinase inhibitors were isolated from human urine by affinity chromatographyon insolubilized carboxymethylpapain followed by ion-exchange chromatography and immunosorption. One objective of the present investigationwas to define the total inhibitory activityof papain-like cysteine proteinases in one biological fluid, urine, by isolating and characterizing the inhibitors which account for all such inhibitory activity.
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