Cathepsin D generates γ-endorphin from β-endorphin

Gráf, László; Kenessey, Ágnes; Patthy, András; Grynbaum, Alice; Marks, Neville; Lajtha, Ábel

doi:10.1016/0003-9861(79)90012-2

Cited by 43 publications

(11 citation statements)

References 17 publications

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“…The hydrolytic activity of the Triton extract on y-endorphin, dynorphin-(1-13). and &endorphin was due to the presence, in addition to the above, of aminopeptidases, carboxypeptidases, cathepsin D, and a neutral endopeptidase (Graf et al, 1979;Burbach et al, 1981).…”

Section: Metal Ejjects Andph Optimum Profilementioning

confidence: 99%

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

Hui¹,

Wang²,

Lajtha³

1983

Biochemistry

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A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.

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Section: Metal Ejjects Andph Optimum Profilementioning

confidence: 99%

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

Hui¹,

Wang²,

Lajtha³

1983

Biochemistry

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“…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.…”

Section: Methodsmentioning

confidence: 99%

“…Sci. USA 76 (1979) 6123 traction step of the human "'-MSH" isolation procedure was performed at pH 5.5 (14), a pH close to the pH optimum of cathepsin D (17,24).…”

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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“…The specificity and susceptibility of the pituitary extract to pepstatin were found to be the same as to bovine pituitary cathepsin D. This last-named enzyme is also capable of splitting the Leu 77-Phe 78 bond of beta'-endorphin to give gamma-endorphin. Graf et al (225,226) therefore suggested that cathepsin D is at least a major factor in the generation of medium-sized endorphins from betaendorphin. When cathepsin D reacts with beta-LPH, however, no intact gamma-endorphin is released, which suggests that beta-endorphin needs first to be produced in another enzymatic reaction; this accords with the pulse-chase experiments of Crine et al (151).…”

Section: Maturation Enzymesmentioning

confidence: 97%

“…Graf et al (225,226) showed that a crude extract of porcine anterior pituitary contains endopeptidase activity capable of splitting the Leu 77-Phe 78 bond of beta-LPH over a pH range of 3-7. The specificity and susceptibility of the pituitary extract to pepstatin were found to be the same as to bovine pituitary cathepsin D. This last-named enzyme is also capable of splitting the Leu 77-Phe 78 bond of beta'-endorphin to give gamma-endorphin.…”

Section: Maturation Enzymesmentioning

confidence: 99%

Chemistry and biosynthesis of pro-opiomelanocortin

Chrétien

Seidah

1981

Mol Cell Biochem

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Studies of lipotropins, melanotropins and endorphins on one hand, and of adrenocorticotropin on the other, has given rise to the concept of a multipotent precursor molecule recently renamed proopiomelanocortin. The preferential sites of cleavage of the precursor to produce its biologically active components are made of pairs of basic amino acid residues as described for the biosynthesis of beta-MSH and pro-insulin. Such structural feature is also found in other pro-hormone molecules. Pulse chase experiments and secretory studies carried out in both anterior and intermediate lobes of rat pituitary glands revealed the transformation of different forms of the precursor into different end-products, the anterior lobe producing preferentially ACTH and beta-LPH while the intermediate produces mainly the alpha-MSH and beta-endorphin. The multiple forms of precursors seem to differ in their carbohydrate content although at least two different gene products are still possible. The presence of similar peptides in the hypothalamus makes it highly probable that neuropeptides are biosynthesized with similar process. Thus the model of beta-LPH precursor, proposed as early as in 1967, is now applicable to the biosynthesis of all other neuropeptides. Major advances in this field are expected in the 1980s.

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Cathepsin D generates γ-endorphin from β-endorphin

Cited by 43 publications

References 17 publications

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

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

Chemistry and biosynthesis of pro-opiomelanocortin

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