Proteolytic processing of farnesylated peptides: assay and partial purification from pig brain membranes of an endopeptidase which has the characteristics of E.C. 3.4.24.15.

The proteolytic processing of farnesylated peptides was investigated with the tritiated C-terminal heptapeptide of mouse N-Ras protein, Propionyl-Gly-Ser-Pro-(farnesyl-Cys)-[3H]Val-Leu-Met, as substrate. Using two rapid and sensitive methods, the peptidase which cleaves the (farnesyl-Cys)-[3H]Val bond was purified more than 100 times from a microsomal fraction of pig brain. The determination of its molecular mass (about 70 kDa) and its pH range for optimum activity (neutral pH) as well as the results obtained with various inhibitors indicate that this [3H]Val-Leu-Met releasing enzyme resembles endopeptidase 24.15: a thiol-dependent zinc metallopeptidase. Parallel and comparative inhibition assays on partially purified enzyme and pure "24.15" confirm this similarity.

Cleavage of farnesylated COOH-terminal heptapeptide of mouse N-ras by brain microsomal membranes: evidence for a carboxypeptidase which specifically removes the COOH-terminal methionine.

Brain microsomal membranes are capable of sequentially removing Met, Leu and Val from a chemically synthesized COOH-terminal heptapeptide (propionyl-Gly-Ser-Pro-(farnesyl-Cys)-Val-Leu-Met) of mouse N-ras protein. The carboxypeptidase generating Met displays maximum activity at neutral pH and shows high affinity for the farnesylated substrate (Km = 73 microM) as compared to its non farnesylated precursor (Km = 600 microM). The results of inhibitor action suggest that the membrane carboxypeptidase is a novel, probably thiol-dependent, serine type peptidase.

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

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.

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.

Synaptosomal degradation of substance P and some other neuropeptides.

Synaptosomes purified from spinal cord and from different rat brain areas exhibit peptide hydrolase activity, cleaving substance P (SP), bradykinin, THRH, LHRH, and neurotensin. The lowest activity for all the peptides tested was found in spinal cord, while the region with the highest degrading activity depended on the substrate: for substance P, it was striatum and cortex; for bradykinin, hypothalamus, and medulla oblongata; for THRH, striatum; for LHRH, midbrain; and for neurotensin, hippocampus. Degradation of substance P takes place at the plasma membrane of synaptosomes. Synaptosome ghosts cleave substance P (pH optimum 7-9, Km-2.5 X 10(-5) M, Vmax-130 nmol . hr-1 . mg protein-1) and also a number of its C-terminal fragments. Effects of the inhibitors show that several different classes of peptidases and proteases are involved in the degradation process. Peptide cleavage represents the probable pathway of synaptosomal inactivation of substance P.

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.

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.

In vitro degradation of substance P: new rapid assay and distribution in rat brain.

An ion exchange batchwise procedure was developed for the estimation of substance P degradation by rat brain fractions using 3H[Nle11]substance P as tracer. With this very sensitive and rapid method an especially high degradative activity was found among others in the microsomal fractions of the striatum.

Hypothalamic cathepsin D: assay and isoenzyme composition.

A sensitive and convenient method of endopeptidase assay using as substrate globin modified with pyridoxal-5-phosphate was used for determination of acid proteinases in bovine hypothalamus separated by isoelectric focusing. The soluble protein fraction of hypothalamus upon elution from Sephadex gave five peaks of proteinase activity at pH 3.2. The properties indicate that these peaks of endopeptidase activity are isoenzyme forms of cathepsin D.

Acid proteinase of hypothalamus. Purification, some properties, and action on somatostatin and substance P.

In a continuing study of the physiological role of protein breakdown in the hypothalamus, acid proteinase from bovine hypothalamus was purified about 1000-fold. The molecular weight of the enzyme was approximately 50,000. Masimal activity against hemoglobin was obtained at pH 3.2-3.5; serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by beta-phenyl pyruvate, or benzethonium Cl, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, substance P, and analogs of substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. The enzyme, most likely cathepsin D, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus.

Beta-cyanoalanine synthase: purification and characterization.

Beta-cyano-L-alanine synthase [L-cysteine hydrogen-sulfide-lyase (adding HCN), EC 4.4.1.9] was purified about 4000-fold from blue lupine seedlings. The enzyme was homoegeneous on gel electrophoresis and free of contamination by other pyridoxal-P-dependent lyases. The enzyme has a molecular weight of 52,000 and contains 1 mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4.7. Its absorption spectrum has two maxima, at 280 and 410 nm. L-Cysteine is the natural primary (amino acid) substrate; beta-chloro- and beta-thiocyano can serve (with considerably lower affinity) instead of cyanide as cosubstrates for cyanoalanine synthase. The synthase is refractory to DL-cycloserine and D-penicillamine, potent inhibitors of many pyridoxal-P-dependent enzymes. Cyanoalanine synthase catalyzes slow isotopic alpha-H exchange in cysteine and in end-product amino acids; the rates of alpha-H exchange in nonreacted (excess) cysteine are markedly increased in the presence of an adequate cosubstrate; no exchange is observed of H atoms in beta-position.