Breakdown of somatostatin and substance P by cathepsin D purified from calf brain by affinity chromotography

Benuck, M.; Grynbaum, Alice; Marks, Neville

doi:10.1016/0006-8993(78)90764-3

Cited by 62 publications

(18 citation statements)

References 15 publications

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“…Isolation of cathepsin D from bovine brain was carried out by a modification of the procedure of Benuck et al (16). The brains were removed within 1 hr of killing and homogenized in 2 vol of 0.05 M sodium citrate, pH 2.7, in an Ultraturrax homogenizer (Janke-Kunkel KG) at 4°C for 40 s. The homogenate was centrifuged at 25,000 X g for 20 min at 4°C and the supernatant fraction was stored at -20°C for later purification.…”

Section: Methodsmentioning

confidence: 99%

Action of cathepsin D on human beta-lipotropin: a possible source of human "beta-melanotropin".

Barát¹,

Patthy²,

Gráf³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Highly purified calf brain cathepsin D (EC 3.4.23.5) selectively splits the LeuO-Phe78 and Ala3-Ala37 peptide bonds of human P-lipotropin. It is suggested that the formation of human "jB-melanotropin" from y-lipotropin, and that of -y-endorphin from f.endorphin, is due to the action of cathepsin D during isolation procedures.Since the discovery of 3-lipotropin (3-LPH) and its isolation from ovine pituitaries (1), our knowledge of the chemistry of lipotropins has steadily grown. The complete amino acid sequences (91 amino acids) of ovine (2-4), porcine (5), human (6), and bovine (7) (14) and subsequently identified as a 22-residue peptide containing four more amino acids than certain other species homologs (8, 15), might have been artifactually formed from human f-LPH during the extraction procedure.The present paper provides evidence for the proposition that lysosomal cathepsin D (EC 3.4.23.5) is one of the proteinases involved in the generation of human ",-MSH" from human 3-LPH. EXPERIMENTAL Isolation of cathepsin D from bovine brain was carried out by a modification of the procedure of Benuck et al. (16). The brains were removed within 1 hr of killing and homogenized in 2 vol of 0.05 M sodium citrate, pH 2.7, in an Ultraturrax homogenizer (Janke-Kunkel KG) at 4°C for 40 s. The homogenate was centrifuged at 25,000 X g for 20 min at 4°C and the supernatant fraction was stored at -20°C for later purification.Pepstatin-Sepharose was prepared as follows: 0.7 mg of pepstatin (The Protein Research Foundation, Osaka, Japan), 0.5 g of AH-Sepharose 4B (Pharmacia), and 16.5 mg of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meto-p-toluenesulfonate (Fluka, Buchs, Switzerland) were mildly agitated in 10 ml of dimethylformamide/water, 1:3 (vol/vol), pH 6, for 16 hr at room temperature. The product was filtered and washed with 50-ml portions of dimethylformamide/water, 1:1 (vol/vol); dimethylformamide/dioxane, 1:1 (vol/vol); 4 M urea; and 0.1 M sodium citrate, pH 3.9/1 M NaCl in this sequence. The pepstatin-Sepharose column (5 cm X 0.7 cm) was prepared and washed alternately with 0.1 M sodium bicarbonate, pH 7.6/1 M NaCl and 0.1 M sodium citrate, pH 3.9/1 M NaCI, equilibrated with the latter solution, and subsequently loaded with 25 ml of the brain extract previously dialyzed against the pH 3.9 solution for 10 hr. After the column was washed with the latter solution, cathepsin D was eluted with 0.1 M sodium bicarbonate, pH 7.6/1 M NaCl, and the pH of the enzyme solution was adjusted to about 4 by addition of 1 M sodium citrate, pH 3.9. Enzyme solutions were kept frozen for later use.Cathepsin D was assayed on porcine f3-LPH (,Bp-LPH) as described (17). Op-LPH and human 3-LPHs (3b-LPHs) were prepared according to Graf and Cseh (18) and Cseh et al. (19,20), respectively.Digestion of f3-LPH with purified cathepsin D was performed in 0.1 M ammonium formate, pH 4, at a ratio of 1 to 1000 (wt/wt) of enzyme to peptide at 37°C. Digestion was terminated by addition of 1 ,uM pepstatin to the incubation mixture.The 13h-LP...

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Section: Methodsmentioning

confidence: 99%

Action of cathepsin D on human beta-lipotropin: a possible source of human "beta-melanotropin".

Barát¹,

Patthy²,

Gráf³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Further work of this group confirmed these data at pH 4.0 and demonstrated that the formation of fl-lipotropin-(1-77) by brain homogenates was due to cathepsin D activity (Gr/tf et al, 1979a). Cathepsin D isolated from bovine brain by affinity chromatography (Benuck et al, 1978a) cleaved the Leu-Phe bond of porcine and human fl-lipotropin (Benuck et al, 1978b;Grfif et al, 1979b;Barfit et al, 1979). At pH 8 porcine fl-lipotropin was degraded only very slowly by brain homogenates.…”

Section: Proteolysis Of Fl-lipotropin By Brain Enzymesmentioning

confidence: 99%

“…Cathepsin D purified from calf brain by affinity chromatography on pepstatin-Sepharose (Benuck et al, 1978a) split initially the Leu-Phe bond of human (residues 75-76) and porcine/~-lipotropin (residues 77-78), forming //-lipotropin-(1-75) and -(1-77) respectively, and /3-endorphin-(18-31) (Gr~if et al, 1979b;Barht et al, 1979). During longer incubation of porcine/~-lipotropin the Ala3Z-Glu 33 peptide bond was cleaved as well by the enzyme preparation.…”

Section: Cathepsin Dmentioning

confidence: 99%

“…The Leu~7-Phe ~8 bond of fl-endorphin is a site sensitive to the action of the purified acid proteinase cathepsin D. Cathepsin D purified from calf brain by affinity chromatography on pepstatin-Sepharose (Benuck et al, 1978a) exclusively cleaved fl-endorphin at the LeulT-Phe 18 bond forming ~-endorphin and fl-endorphin-(18-31) (Gr~tf et al, 1979b). The enzyme displayed activity in the pH range 3.0-7.0 and was completely inhibited by 10 -6 M pepstatin.…”

Section: Purified Enzymes From Brain Tissuementioning

confidence: 99%

“…The enzyme displayed activity in the pH range 3.0-7.0 and was completely inhibited by 10 -6 M pepstatin. Cathepsin D purified from other tissues, such as human pituitaries (Benuck et al, 1978a;Bar,it et al, 1979) and bovine spleen, obtained as a commercial preparation (Burbach et al, 1980b), cleaved the Leu-Phe bond offl-endorphin or fl-LPH similarly (see also section 3.1.2. ).…”

Section: Purified Enzymes From Brain Tissuementioning

confidence: 99%

See 2 more Smart Citations

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

Burbach¹

1984

Pharmacology & Therapeutics

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Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin

Karlsson¹,

Nyberg²

1998

J. Mol. Recognit.

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An enzyme activity capable of hydrolysing the neuroactive undecapeptide substance P (SP) between its Phe7-Phe8 residues was purified from the membrane-bound fraction of human spinal cords. The enzyme preparation yielded was compared with a previously described SP-hydrolysing enzyme from human cerebrospinal fluid (CSF) with regard to inhibition profile, protein chemical properties and kinetics. In addition, the results were compared with those of bovine pancreatic chymotrypsin (a serine protease that cleaves the carboxy-terminal side preferentially at hydrophobic amino acids). The SP peptidase activity was extracted from human spinal cords with 1% Triton X-100 in 20 mM Tris-HCI pH 7.8. After ion exchange chromatography (DEAE-Sepharose) where the enzyme activity was separated from other proteins by gradient elution, the pooled enzyme fraction was further purified by molecular sieving (Sephadex G-50). The enzyme activity was finally recovered by HPLC molecular sieving (Superdex 75 HR 10/30) using a new preparative system, AKTA-purifier, controlled by UNICORN software version 2.20.

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Breakdown of somatostatin and substance P by cathepsin D purified from calf brain by affinity chromotography

Cited by 62 publications

References 15 publications

Action of cathepsin D on human beta-lipotropin: a possible source of human "beta-melanotropin".

Action of cathepsin D on human beta-lipotropin: a possible source of human "beta-melanotropin".

Action of proteolytic enzymes on lipotropins and endorphins: Biosynthesis, biotransformation and fate

Purification of substance P endopeptidase (SPE) activity in human spinal cord and subsequent comparative studies with SPE in cerebrospinal fluid and with chymotrypsin

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