Production of (S)-.ALPHA.-benzylmalic acid, inhibitor of carboxypeptidase A by actinomycetes.

The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.

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The protease phenotype and crystalline nature of the granules of intraepithelial mast cells in Trichinella spiralis-infected mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140464 ◽

1995 ◽

Vol 53 ◽

pp. 818-819

Author(s):

D.S. Friend ◽

N. Ghildyal ◽

M.F. Gurish ◽

K.F. Austen ◽

R.L. Stevens

Keyword(s):

Small Intestine ◽

Mast Cells ◽

Epithelial Cells ◽

Lamina Propria ◽

Trichinella Spiralis ◽

Intestinal Epithelial ◽

Carboxypeptidase A ◽

C3h Mice ◽

Granule Morphology ◽

Crystalline Nature

Trichinella spiralis induces a profound mastocytosis and eosinophilia in the small intestine of the infected mouse. Mouse mast cells (MC) store in their granules various combinations of at least five chymotryptic chymases [designated mouse MC protease (mMCP) 1 to 5], two tryptic proteases designated mMCP-6 and mMCP-7 and an exopeptidase, carboxypeptidase A (mMC-CPA). Using antipeptide, protease -specific antibodies to these MC granule proteases, immunohistochemistry was done to determine the distribution, number and protease phenotype of the MCs in the small intestine and spleen 10 to >60 days after Trichinella infection of BALB/c and C3H mice. TEM was performed to evaluate the granule morphology of the MCs between intestinal epithelial cells and in the lamina propria (mucosal MCs) and in the submucosa, muscle and serosa of the intestine (submucosal MCs).As noted in the table below, the number of submucosal MCs remained constant throughout the study. In contrast, on day 14, the number of MCs in the mucosa increased ~25 fold. Increased numbers of MCs were observed between epithelial cells in the mucosal crypts, in the lamina propria and to a lesser extent, between epithelial cells of the intestinal villi.

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