Protein inhibitors of proteinases from brain.
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T Akopyan
Publications and source records attributed to T Akopyan.
Pyridoxal-5'-phosphate derivatives of substance P: preparation, spasmogenic activity, and degradation by human plasma.
Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) were prepared by chemical modification of the native peptide by pyridoxal-5'-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the epsilon-amino group of the Lys residue [epsilon-(P-pxy)-SP] and in the other the epsilon-amino group of the Lys residue and also the N-terminal amino group [alpha, epsilon-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. epsilon-(P-pxy)-SP and alpha, epsilon-di-(P-pxy)-SP have spasmogenic activity with ED50 of 1.8 X 10(-9) and 4 X 10(-9) M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of epsilon-(P-pxy)-SP and 0.5 pmol/ml of alpha, epsilon-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.
Animals↗
Partial purification and some properties of a bovine brain trypsin inhibitor.
Using hemoglobin modified by pyridoxal 5'-phosphate as substrate, a trypsin inhibitor from bovine brain was purified by extraction at pH 4.5, ion-exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100 and isoelectric focusing. On a column of Sephadex G-100 the inhibitor exhibited a molecular mass of 78 kDa. The iso-electric point of the inhibitor was 4.3-4.4. The dissociation constant (Ki) for the complex of bovine trypsin and brain inhibitor was estimated to be 3.7 X 10(-10)M as tested with a protein substrate, and 2.4 X 10(-10)M when tested with a synthetic substrate. During purification two other brain trypsin inhibitors were detected.
Animals↗
Cathepsin D from human brain: purification and multiple forms.
Cathepsin D was purified about 1000-fold from human brain cortex by a procedure involving ammonium sulfate fractionation (30-70%), Sephadex G-75 chromatography, affinity chromatography on pepstatin-Sepharose and isoelectric focusing. The enzyme was assayed fluorometrically at pH 3.2, the substrates used were globin or haemoglobin modified with pyridoxal-5'-phosphate. 6 multiple forms of cathepsin D were resolved in the isoelectric focusing step with pI values 4.4, 4.8, 5.3, 6.2, 6.5 and 6.8. Km of pyridoxal-globin and pyridoxal-haemoglobin for all 6 multiple forms is 1.8-2.0 X 10(-5) M and 1.3 to 4 X 10(-6) M, respectively, and Ki of pepstatin is 2-4 X 10(-9) M. Gel filtration of the multiple forms on Sephadex G-100 column showed that each has a molecular weight of about 50 000. Human brain cathepsin D has a pH optimum of 3.2 with a smaller second optimum at pH 4.0 (pyridoxal-haemoglobin being used as substrate). All the multiple forms have the same pH-dependence curve. On SDS-polyacrylamide gel electrophoresis the purified enzyme produced 3 bands approximately corresponding to Mr 50 000, 35 000 and 15 000. Study of the breakdown of substance P and its C-terminal heptapeptide by cathepsin D shows that cleavage occurs at the Phe-Phe linkages of both substrates tested.
Brain↗