Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold iinmunocytochemical method. Duodenase exhibits trypsin-like and chyinotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be a-N-tosylglycylprolyllysine 4-nitroanilide (kJK,,, of 35 000 M-' s-'), a-N-succinylthreonylprolyllysine 4-nitroanilide (kc4,/K1,! of 18 000 M-' s-') and a-N-serylprolyllysine 4-nitroanilide (k,JK,, of 2600 M-' s-I), all of which contain the P I -P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9 -PS'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (K,,, of 87 pM; k,;,, of 1.4 s-I; k,,,/K,,, of 16000 M-' s-I). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron Xrays to 0.2-nm resolution.

Duodenase, a serine proteinase from bovine Brunner's (duodenal) glands that was predicted to be a natural activator of enteropeptidase zymogen, cleaves and activates recombinant single-chain bovine proenteropeptidase (k cat /K m = 2700 M 31 s 31 ). The measured rate of proenteropeptidase cleavage by duodenase was about 70-fold lower compared with the rate of trypsin-mediated cleavage of the zymogen. The role of duodenase is supposed to be the primary activator of proenteropeptidase maintaining a certain level of active enteropeptidase in the duodenum. A new scheme of proteolytic activation cascade of digestive proteases is discussed.z 2000 Federation of European Biochemical Societies.

We report on a miniature spectrometer with a volume of 0.135 cm(3) and dimensions of 3x3x11 mm, mounted directly on the surface of a CCD sensor. The spectrometer is formed by two flat diffraction gratings that are designed to perform both the dispersion and imaging functions, eliminating the need for any spherical optics. Two separate parts of the device were fabricated with the single-mask 1 mum lithography on a single glass wafer. The wafer was diced and the device was assembled and directly mounted onto a CCD sensor. The resolution of 3 nm, spectral range of 450 to 750 nm and the optical throughput of ~9% were measured to be in a complete agreement with the model used for the development of the device.

In this report we propose a new approach to classification of serine proteases of the chymotrypsin family. Comparative structure-function analysis has revealed two main groups of proteases: a group of trypsin-like enzymes and graspases (granule-associated proteases). The most important structural peculiarity of graspases is the absence of conservative "active site" disulfide bond Cys191-Cys220. The residue at position 226 in the S1-subsite of graspases is responsible for substrate specificity, whereas the residue crucial for specificity in classical serine proteases is located at position 189. We distinguish three types of graspases on the base of their substrate specificity: 1) chymozymes prefer uncharged substrates and contain an uncharged residue at position 226; 2) duozymes possess dual trypsin-like and chymotrypsin-like specificity and contain Asp or Glu at 226; 3) aspartases hydrolyze Asp-containing substrates and contain Arg residue at 226. The correctness of the proposed classification was confirmed by phylogenic analysis.

A high dispersion, multichannel, visible spectrometer has been developed for the ISTTOK tokamak, aiming at measuring the evolution of both the edge ion temperature and plasma poloidal velocity, by analysis of a CIII spectral line. It is based on a double monochromator configuration with two high ruling densities (2700 l/mm) and 1 m focal length concave diffraction gratings, a commercial double microchannel plate image intensifier, a fast (f/#=1) relay lens built with two high diameter condensers in a back-to-back configuration, and a 2048 element photodiode charge coupled device optical multichannel analyzer. Laboratory tests have shown that a high dispersion has been achieved (≈1.65 Å/mm), while keeping good temporal resolution (5 ms). Typical values measured for ion temperature (from Doppler broadening) were Ti≅25 eV while for poloidal velocity (from Doppler shift) vθ≅6 km/s.