We point out that human low-Mr kininogen contains three cystatin-like sequences, rather than two, as had previously been thought. The protein was purified by affinity chromatography on carboxymethyl-papain-Sepharose, and subjected to limited proteolysis by trypsin and chymotrypsin. Fragments were isolated, and three corresponding to the individual cystatin-like domains were identified. By comparison with the known amino acid sequence of the protein they were numbered 1 to 3 from the N-terminus. Domain 1 was not found to have any inhibitory activity for cysteine proteinases, which is consistent with the absence of residues that are highly conserved in inhibitors of the cystatin superfamily, and have previously been suggested to be essential for activity. Domain 2 was a good inhibitor of chicken calpain, and also papain and cathepsin L. Domain 3 showed negligible inhibition of calpain, but inhibited papain and cathepsin L strongly. The probable arrangement of disulphide bonds in the heavy chain of low-Mr kininogen is deduced from the homology with the cystatins and other evidence contained in the present paper.
The tissue distribution, developmental changes, and the vitamin D dependence in the rat of two calcium-binding proteins [CaBPs, 28,000 and 10,000 Mr (28 and 10 K)] were examined. The radioimmunoassays used employed specific antibodies to either the human cerebellar CaBP (28 K protein) or to the smaller rat intestinal CaBP (10 K protein). The assay for the 28 K CaBP may be used to detect this protein in a number of mammalian species and tissues, whereas the 10 K CaBP assay appears to be specific only for the rat intestinal CaBP. This report demonstrates that the tissue distribution of the two CaBPs is different in the rat. High levels of the 28 H protein were found in the cerebellum and kidney, whereas the smaller CaBP was concentrated in the duodenum, jejunum, and cecum. Many other organs and tissues contained small quantities of both CaBPs. Developmental studies indicated some variability in the concentration of the CaBPs. Duodenum, kidney, and cerebellum all contained small amounts of one of the CaBPs prior to birth. Adult levels in all three tissues were already reached at 30 postpartum days. Levels of both CaBPs began to decline in rats older than 2 mo. The vitamin D dependence appeared to reflect cell turnover in that the duodenal and kidney CaBPs showed a vitamin D dependence not observed for the cerebellar protein.
A Ca2+-activated cysteine proteinase (calpain II) was purified from chicken gizzard smooth muscle by use of isoelectric precipitation, (NH4)2SO4 fractionation, chromatography on DEAE-Sepharose CL-6B, Reactive-Red 120-agarose and Mono Q. The apparent second-order rate constants for the inactivation of calpain by a series of structural analogues of L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-guanidino)butane (E-64) were determined. The fastest rate of inactivation was observed with L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-benzyloxy-carbonylamino)buta ne. It was possible to determine the active-site molarity of solutions of calpain by titration with E-64. When incubated with Ca2+, calpain underwent several steps of intermolecular limited proteolysis, via multiple pathways, followed by a slower loss of enzymic activity. The proteolytic steps preceding the loss of activity did not affect the rates of reaction of calpain with E-64 analogues.
Specific antibodies raised against a human 28 000 dalton cerebellar calcium-binding protein (CaBP) were used in an immunocytochemical study during development of the rat cerebellum. Both light and electron microscopy showed (1) that labelling was entirely restricted to the Purkinje cells, (2) that it appeared very early in Purkinje cell development, (3) that the entire cell was labelled from the tip of the smallest dendrites to the axonal terminals, and (4) that with increasing age, the immunoreaction appeared to be progressively restricted to the cell and organelle membranes.
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