Metalloendopeptidase QG. Isolation from Escherichia coli and characterization.

A new proteinase, which preferentially cleaves the Gln-Gly bond, was isolated from Escherichia coli. Because of this narrow specificity, the enzyme was called metalloendopeptidase QG. The proteinase is a monomer and consists of a single polypeptide chain of Mr 67,000, which is significantly smaller than the other known metalloendopeptidases of E. coli. It is found in the cytoplasm, but not in the periplasm. The enzyme cleaves the substrate benzyloxycarbonyl-Gln-Gly-Pro 2-naphthylamide between the glutamine and glycine residues, as well as its extended homologues including a nonapeptide, but it does not hydrolyse either the oxidized A and B chains of insulin or azo-casein. The pH-dependence of substrate hydrolysis gives a bell-shaped curve with pK1 = 6.6 and pK2 = 8.8. The metallopeptidase is inhibited in Tris and imidazole buffers, the basic components of which are presumably liganded to the essential Zn2+ ion. 2-Aminobenzoyl-Gln-Gly-Pro 2-naphthylamide, designed as a fluorescent substrate for the metallopeptidase, proved to be a strong inhibitor. Bestatin, an inhibitor of aminopeptidases in the micromolar concentration range, inhibits the metalloendopeptidase only in the millimolar concentration range. Captopril, the widely used inhibitor of angiotensin-converting enzyme, is a fairly good inhibitor of the metalloendopeptidase. The simplest inhibitor that can be used to protect recombinant proteins from degradation by the metalloendopeptidase may be EDTA, which is effective at low millimolar concentration.

Disulfide bond formation between the active-site thiol and one of the several free thiol groups of chymopapain.

Chymopapain, a cysteine protease of papaya latex, has been purified with the use of fast protein liquid chromatography. Two homogeneous fractions were analyzed for thiol content and thiol reactivity. It was found that peak 1 and peak 2 contained two and three thiol groups, respectively, per mole of enzyme. This result is inconsistent with the general belief that chymopapain contains one essential and one nonessential thiol group and suggests that a significant portion of the thiol groups was oxidized in the previous preparations. Such an oxidation can account for some of the inconsistent results reported in the literature. An irreversibly oxidized nonessential thiol group may modify the catalytic function of chymopapain especially if it is close to the active site. That one thiol group resides indeed in the vicinity of the essential thiol group is clearly demonstrated by the biphasic reactions of chymopapain with disulfide compounds such as 2,2'-dipyridyl disulfide and 5,5'-dithiobis(2-nitrobenzoate). In the first step of these reactions a mixed disulfide is formed between the enzyme and the reactant, which is followed by a first-order, intramolecular reaction leading to the liberation of the second half of the disulfide compound. Furthermore, on addition of one Hg2+ ion, 2 mol of thiol group, one essential and one nonessential, disappears concomitantly. Formation of a disulfide bond between the catalytically competent thiol group and another free thiol group of chymopapain under physiological conditions may be of regulatory importance.

Examination of the uterine cervix by ultrasound in normal and pathologic pregnancy.

New possibilities of examination of the uterine cervix are provided by sonography in normal and pathologic pregnancy. Basic data of the ultrasonographic anatomy of the non pregnant uterine cervix is presented first: the length of the cervix as well as the diameters at the levels of external and internal os. These data are compared then with those in normal, physiologic pregnancy, and contrasted with those obtained in cases of incompetent cervix. In this group shortening of the cervical length, opening of the internal os and the funnel, or hour-glass-like herniation of the fetal membranes were characteristic findings. The method seems to be suitable for the assessment of the effectivity of cerclage operations for cervical incompetence.

Antenatally diagnosed hygroma colli.

A case of prenatally diagnosed hygroma colli is presented. Prenatal diagnosis, characteristic ultrasonic signs as well as problems of differential diagnosis are discussed. The importance of non-elective routine ultrasound screening in the early detection of fetal congenital malformations is emphasized.

Study on the role of tyrosine side-chains at the active centre of emulsin beta-D-glucosidase.

The role of exposed tyrosine side-chains in enzyme-catalyzed reaction by sweet almond emulsin beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) has been studied using N-acetylimidazole as the specific reagent. The changes in activity, binding affinity and kinetic parameters (Km, V) as a result of acetylation of the phenolic hydroxyl groups have been determined. The acetylation increased the Km values of both beta-glucosidase and beta-galactosidase activities, whereas V remained unchanged. Similarly, the binding affinity for immobilized phenyl beta-D-glucopyranoside decreased appreciably. After the removal of the acetyl groups the enzyme regained 96% of the original activity. It is concluded that the tyrosine moieties, located in the active centre of the enzyme, have both glucoside and galactoside binding functions.