Richard E. Galardy

Low Molecular Weight Inhibitors in Corneal Ulcerationa

RICHARD E. GALARDYP MARIE E. CASSABONNE, C A R L A ” E GIESE, JAMES H. GILBERT, FRANCE LAPIERRE, HENRY LOPEZ, MARY E. SCHAEFER, ROBERT STACK, MICHAEL SULLIVAN, BRENT SUMMERS, ROB TRESSLER, DAVE TYRRELL, AND JENNIFER WEEC; SCOTT D. ALLEN AND JOHN J. CASTELLO@; JOHN P. BARLETTA AND GREGORY S. SCHULTZe; LEONARD0 A. FERNANDEZf; SUSAN FISHER AND TIAN-YI CUF; HARALD G. FOELLMERh; DAMIAN GROBELNY’; AND WALTER M. HOLLERANJ

The intramembranous segment of human erythrocyte glycophorin A

Abstract The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work ( J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973 , Biochem. Biophys. Res. Commun. , 49 , 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.

Inhibition of angiotensin converting enzyme with Nα-phosphoryl-L-alanyl-L-proline and Nα-phosphoryl-L-valyl-L-tryptophan

Summary The inhibition constants (Ki) and modes of inhibition have been determined for Nα-phosphoryl-L-alanyl-L-proline and Nα-phosphoryl-L-valyl-L-tryptophan against rabbit pulmonary angiotensin converting enzyme (dipeptidyl carboxpeptidase, E.C. 3.4.15.1). Phosphorylalanylproline and phosphorylvalyltryptophan are noncompeptitive inhibitors of the hydrolysis of hippuryl-L-histidyl-L-leucine in 100 mM phosphate buffer at pH 8.3 with Kis of 9 nM and 51 nM respectively. In 50 mM Tris-hydrochloride buffer at pH 7.5, however, phosphorylalanylproline is a competitive inhibitor of the hydrolysis of hippuryl-L-histidyl-L-leucine with a Ki of 1.4 nM, and thus is one of the most potent inhibitors known for this enzyme.

19F nuclear magnetic resonance spectroscopy study of the complex of 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid and carboxypeptidase A

(dl)-2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is a strong transition state analog inhibitor of the zinc protease carboxypeptidase A. 19F NMR spectroscopy of the aqueous solution of this inhibitor shows the hydrate of the ketone carbonyl to be the major species, with a shift of -9.95 ppm. As the pH is varied from 4.9 to 13.1, a 1.53 ppm downfield shift occurs, giving a pK alpha of 11.10. When excess inhibitor is added to the enzyme, a new, bound peak appears at -8.84 ppm, in addition to the free hydrate peak. Spectra taken at pHs from 4.90 to 9.15 show no change in the position of the bound resonance; from 9.15 to 12.15, a 0.26 ppm upfield shift occurs. The interpretation is that the monoanion of the hydrate is the form that binds to the enzyme.

A metabolite of aspartame inhibits angiotensin converting enzyme

Aspartame (L-aspartyl-L-phenylalanine methyl ester, is a widely used artificIal sweetener. In humans and other animals aspartame is initially hydrolyzed to L-aspartyl-L-phenylalanine by intestinal esterases. L-Aspartyl-L-phenylalanine inhibits angiotensin converting enzyme purified from rabbit lungs with a Ki of 11 +/- 2 microM, equipotent to the IC50 of 12 microM for 2-D-methyl-succinyl-L-proline which has been reported to be an orally active antihypertensive agent in rats. Thus the possibility exists that L-aspartyl-L-phenylalanine inhibits angiotensin converting enzyme in humans consuming large quantities of aspartame. Both aspartame itself and the diketopiperazine formed from it, 3-carboxymethyl-6-benzyl-2,5-diketopiperazine, are weak inhibitors with Kis greater than 1 mM.

The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro is a tetrahedral transition state analog inhibitor of C. histolyticum collagenase

The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro [(4-13C-5-cinnamido-4-oxo-6-phenylhexanoyl)-Pro-Pro 2] competitively inhibits a mixture of collagenases from Clostridium histolyticum with a Ki of 40 +/- 6 nM. 13C-nmr spectroscopy of the ketone in the presence of this collagenase shows a bound 13C resonance at 102.6 ppm and the resonance of the free ketone at 212 ppm. Ketone alone shows no trace (less than 0.5%) of a resonance in the region around 100 ppm. The bound resonance is displaceable by another competitive inhibitor. This ketone is thus a transition state analog which is rehybridized from trigonal planar to tetrahedral upon binding to collagenase.

Folding pathways for the glycophorin transmembrane fragment

The folding of the membrane-spanning segment (residues 73-94) of glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, appears to proceed in vitro in a pathway-dependent manner. The secondary structure of a synthetic polypeptide that comprises this fragment has been studied by circular dichroism (CD) spectroscopy. In both detergent and small unilamellar vesicles, the polypeptide is composed of only a small amount of helical secondary structure. However, if the fragment is first dissolved in trifluoroethanol (TFE) and then transferred to a detergent environment, the resulting structure is predominantly a-helical. The converse is not true. Furthermore, once it has been exposed to trifluoroacetic acid (TFA), the fragment loses its ability to attain a helical conformation, even upon exposure to TFE.