Cystatins from bovine brain: Purification, some properties, and action on substance P degrading activity

Abstract

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80 degrees C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. Ki values for the complexes of papain and the inhibitors were estimated to be 2.8 x 10(-10) M for HMM-cystatin and 1.3 x 10(-9) M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.

The Role of Cystatin C in Cerebral Amyloid Angiopathy and Stroke: Cell Biology and Animal Models

“…Enhanced cystatin C expression was observed in response to injury, including facial nerve axotomy (113), perforant path transections (181), and hypophysectomy (83), in morphologically degenerative CA1 pyramidal neurons and reactive astrocytes of hippocampus following transient forebrain ischemia (70,122,129,180) and after induction of epilepsy (10, 66, 100). It was proposed that cystatin C might facilitate the establishment of an axonal growth-promoting environment by protecting neurite growthassociated molecules from degradation by cysteine proteases in the deafferented hippocampus (6,135). Oxidative stress also stimulates an increase in cystatin C expression in cultured neurons (120,121) and in cerebral microvascular smooth muscle cells (170), suggesting a role in regulation of apoptosis.…”

Genetics of epilepsy: Epilepsy Research Foundation workshop report

“…It may also have an effect on inflammatory response by modulating the migration of inflammatory cells. Cystatin C also prevented the degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes (Aghajanyan et al 1988). The antibacterial effect of the enzymatically active cystatin C amino acid sequence has been proposed to be independent of the proteinase inhibitory effect in the regulation of inflammatory response (data from peripheral tissues).…”

Purification and Characterization of High Molecular Mass and Low Molecular Mass Cystatin from Goat Brain

“…Cystatins have been purified and characterized from various sources including bovine muscle [19], goat kidney [20], human spleen [21], human liver [22], amyloid fibrils [23], human placenta [24], sheep plasma [25], skin of Atlantic Salmon [26] and bovine brain [27].…”