• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Apr 1, 1976; 155(1): 117–125.
PMCID: PMC1172808

Interaction of human cathepsin D with the inhibitor pepstatin.

Abstract

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abu-Erreish GM, Peanasky RJ. Pepsin inhibitors from Ascaris lumbricoides. Isolation, purification, and some properties. J Biol Chem. 1974 Mar 10;249(5):1558–1565. [PubMed]
  • Aoyagi T, Kunimoto S, Morishima H, Takeuchi T, Umezawa H. Effect of pepstatin on acid proteases. J Antibiot (Tokyo) 1971 Oct;24(10):687–694. [PubMed]
  • Barrett AJ. Cathepsin D. Purification of isoenzymes from human and chicken liver. Biochem J. 1970 Apr;117(3):601–607. [PMC free article] [PubMed]
  • Barrett AJ. Human cathepsin B1. Purification and some properties of the enzyme. Biochem J. 1973 Apr;131(4):809–822. [PMC free article] [PubMed]
  • Barrett AJ, Dingle JT. The inhibition of tissue acid proteinases by pepstatin. Biochem J. 1972 Apr;127(2):439–441. [PMC free article] [PubMed]
  • Castellino FJ, Barker R. Examination of the dissociation of multichain proteins in guanidine hydrochloride by membrane osmometry. Biochemistry. 1968 Jun;7(6):2207–2217. [PubMed]
  • Dingle JT, Barrett AJ, Weston PD. Cathepsin D. Characteristics of immunoinhibition and the confirmation of a role in cartilage breakdown. Biochem J. 1971 Jun;123(1):1–13. [PMC free article] [PubMed]
  • Dingle JT, Poole AR, Lazarus GS, Barrett AJ. Immunoinhibition of intracellular protein digestion in macrophages. J Exp Med. 1973 May 1;137(5):1124–1141. [PMC free article] [PubMed]
  • DIXON M. The effect of pH on the affinities of enzymes for substrates and inhibitors. Biochem J. 1953 Aug;55(1):161–170. [PMC free article] [PubMed]
  • Ferguson JB, Andrews JR, Voynick IM, Fruton JS. The specificity of cathepsin D. J Biol Chem. 1973 Oct 10;248(19):6701–6708. [PubMed]
  • Goodwin TW, Morton RA. The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem J. 1946;40(5-6):628–632. [PMC free article] [PubMed]
  • GREEN NM. Kinetics of the reaction between trypsin and the pancreatic trypsin inhibitor. Biochem J. 1957 Jul;66(3):407–415. [PMC free article] [PubMed]
  • GREEN NM, WORK E. Pancreatic trypsin inhibitor. II. Reaction with trypsin. Biochem J. 1953 May;54(2):347–352. [PMC free article] [PubMed]
  • Hall TC, Cocking EC. High-efficiency liquid-scintillation counting of 14C-labelled material in aqueous solution and determination of specific activity of labelled proteins. Biochem J. 1965 Sep;96(3):626–633. [PMC free article] [PubMed]
  • Inouye K, Fruton JS. The inhibition of pepsin action. Biochemistry. 1968 May;7(5):1611–1615. [PubMed]
  • Johnson CH, Knowles JR. The binding of inhibitors to alpha-chymotrypsin. Biochem J. 1966 Oct;101(1):56–62. [PMC free article] [PubMed]
  • Keilová H, Tomásek V. Effect of pepsin inhibitor from Ascaris lumbricoides on cathepsin D and E. Biochim Biophys Acta. 1972 Oct 12;284(2):461–464. [PubMed]
  • Kunimoto S, Aoyagi T, Nishizawa R, Komai T, Takeuchi T. Mechanism of inhibition of pepsin by pepstatin. II. J Antibiot (Tokyo) 1974 Jun;27(6):413–418. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Lundblad RL, Stein WH. On the reaction of diazoacetyl compounds with pepsin. J Biol Chem. 1969 Jan 10;244(1):154–160. [PubMed]
  • McKown MM, Workman RJ, Gregerman RI. Pepstatin inhibition of human renin. Kinetic studies and estimation of enzyme purity. J Biol Chem. 1974 Dec 25;249(24):7770–7774. [PubMed]
  • Morishima H, Takita T, Aoyagi T, Takeuchi T, Umezawa H. The structure of pepstatin. J Antibiot (Tokyo) 1970 May;23(5):263–265. [PubMed]
  • Sapolsky AI, Woessner JF., Jr Multiple forms of cathepsin D from bovine uterus. J Biol Chem. 1972 Apr 10;247(7):2069–2076. [PubMed]
  • Takahashi K, Mizobe F, Change WJ. Inactivation of acid proteases from Rhizopus chinensis, Aspergillus saitoi and Mucor pusillus, and calf rennin by diazoactylnorleucine methyl ester. J Biochem. 1972 Jan;71(1):161–164. [PubMed]
  • Takahashi K, Chang W, Ko J. Specific inhibition of acid proteases from brain, kidney, skeletal muscle, and insectivorous plants by diazoacetyl-DL-norleucine methyl ester and by pepstatin. J Biochem. 1974 Oct;76(4):897–899. [PubMed]
  • Tang J. Specific and irreversible inactivation of pepsin by substrate-like epoxides. J Biol Chem. 1971 Jul 25;246(14):4510–4517. [PubMed]
  • Umezawa H, Aoyagi T, Morishima H, Matsuzaki M, Hamada M. Pepstatin, a new pepsin inhibitor produced by Actinomycetes. J Antibiot (Tokyo) 1970 May;23(5):259–262. [PubMed]
  • Woessner JF., Jr Pepstatin inhibits the digestion of hemoglobin and protein-polysaccharide complex by cathepsin D. Biochem Biophys Res Commun. 1972 May 26;47(4):965–970. [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...