The fungus Tritirachium album Limber was grown by submerged fermentation, investigating the conditions for maximal secretion of proteases into the medium. Enzyme secretion starts when the stationary phase of growth is reached, and when the culture medium is depleted of glucose and amino acids, suggesting a catabolite repression of the enzyme.The main proteolytic enzyme was named proteinase with respect to its keratin hydrolyzing activity. It was isolated from the culture medium and purified by crystallization and chromatography on DEAE-Sephadex and Sephadex G-75. Homogeneity was ascertained by disc gel electrophoresis and by isoelectric focusing. The molecular weight of proteinase K was determined by gel filtration to be 18500 f 500, the isoelectrice point was pH 8.9. The enzyme was shown to be a serine protease by inactivation with diisopropyl phosphofluoridate and phenylmethylsulfonyl fluoride. It displayed strong proteolytic activity on denatured but also on native proteins as demonstrated by the rapid inactivation of bovine ribonuclease. The pH-optimum was in the range between pH 7.5-12.0. A specificity of the enzyme for peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids was observed.Screening for microorganisms with proteolytic activity towards native proteins we found a strain of the mold Tritirachium album which secrets a highly active mixture of proteases along with an aminopeptidase into the culture medium. The isolation of proteases from this organism seemed of interest to us because the organism can grow on native keratin as sole carbohydrate and nitrogen source.In this paper the cultivation of Tritirachium album and the purification and some properties of the main protease will be described. This enzyme was named "proteinase K" with respect to its keratin hydrolyzing activity. A characteristic property of the enzyme is its ability to hydrolyze native proteins. This is the first Abbreviations. Ac, acetyl-; Ac-Tyr-OEt, acetyl-Ltyrosine ethyl ester; Bz-Arg-OEt, benzoyl-L-arginine ethyl ester; 2' : 3'-CMP, 2' : 3'-cyclic cytidine monophosphate; EDTA, ethylenediamine tetraacetate; Tos-Lys-CHzC1, l-chloro-3-tosylamido-7-amino-2-heptanone (tosyl lysylchloromethyl ketone); Tos-Phe-CHzC1, L-( I -tosylamido-2-pheny1)ethyl chloromethyl ketone; Z-, benzyloxycarbonyl-; -Nan, 4-nitroanilide.Enzymes. Creatine kinase (EC 2.7.3.2); lactate dehydrogenase (EC 1.1.1.27); malate dehydrogenase (EC 1.1.1.37); ribonuclease (EC 3.1.4.22).Definition. One Anson-U is defined as that amount of enzyme which, under standard conditions, liberates one millimole of Folin-positive amino acids (calculated as tyrosine) per min.report on the purification of a fungal protease from the genus Tritirachium. MATERIALS AND METHODSAll reagents, enzymes and enzyme substrates were products of E. Merck (Darmstadt). OrganismTritirachium album Limber (Merck strain No. 2429) was isolated from soil manured with horn chips. The strain is deposited at the Centraalbureau voor Schimmelcultures at Baarn (CBS no. 747.69). C...

New higher precision measurements of the hyperfine Zeeman transitions in the ground state of muonium have been performed with use of the high-stopping-density surface \i + beam at the Clinton P 0 Anderson Meson Physics Facility. The results are Ay = 4 463-302.88(16) kHz (0.036 ppm) and n^/np^ 3.183 3461(11) (0.36 ppm). The current theoretical value of Ay agrees well with experiment within the 0.77-ppm error of Ay t^eor , which is due principally to inaccuracy in evaluation of the nonrecoil radiative correction term. The most precise current value of m n /m e is obtained from our value of \ij\x p ,

In 1996 131.5 million daily doses of preparations containing extracts of Hypericum perforatum L. were prescribed in Germany for treating mild to moderately severe depressive disorders. New pharmacological and clinical results focus on hyperforin as the main active ingredient of the drug. Hyperforin (C35H52O4) is one of the main components (2-4%) of the dried herb Hypericum perforatum L. It was isolated after six consecutive steps: extraction of deep-frozen blossoms (-20 degrees C) with n-hexane by means of an Ultra Turrax at room temperature; separation of lipophilic substances on a silica gel column; purification of the relevant fraction by preparative HPLC; evaporation of the mobile phase under reduced pressure; removal of the remaining water by freeze-drying; and storage of hyperforin at -20 degrees C under nitrogen. The identity and purity of the isolated substance were determined by high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC) with diode-array and ultraviolet detection (DAD and UV), Fourier-transformed infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy, and liquid chromatography coupled with positive-ion electrospray-ionization tandem mass spectrometry (LC-ESI(+)-MS-MS). By use of these methods the purity of hyperforin was shown to be >99.9%. Peroxides present at each step of the isolation were detected by titration and by means of Merckoquant analytical peroxide test-strips. Elimination of the peroxides and stabilization of hyperforin was achieved by consistent protection from oxidation-the mobile phases were protected by use of ascorbic acid; evaporation and freeze-drying were performed under nitrogen; and the mobile phase used for preparative HPLC was sparged with helium. Stability testing was performed by HPLC-the samples were stored at -30 degrees C in a normal atmosphere and at -20, 4, and 20 degrees C in a normal atmosphere or under nitrogen. Results were compared with those obtained after storage under liquid nitrogen (-196 degrees C). Because of its high sensitivity to oxidation, hyperforin was more stable under nitrogen under all test conditions. There was no statistically significant difference between results obtained after 8 months at -20 degrees C under nitrogen or at -30 degrees C under a normal atmosphere and those from the reference sample stored under liquid nitrogen (-196 degrees C). Despite this, because of the tendency of hyperforin to degrade, long-term storage at -70 degrees C under nitrogen is recommended.

Abstract. Microwave transitions between the Zeeman split doublet states 22P1/2 and 22P3/2 of 7El have been measured using the optical double resonance method. The fine structure separation was determined to be vfs= 10.053.184(58) MHz. The hyperfine structure was well resolved and led to considerably improved precision of the diagonal coupling constants al/2 =45.914(25) MHz, a3/2 = -3.055(14) M Hz, b = -0.22l(29) MHz. Furthermore, the contact, spin dipolar and orbital magnetic hyperfine constants are evaluated using additional results from level crossing experiments, which are more sensitive on the nondiagonal magnetic hyperfine constant a3/2, 1/2. The results are in good agreement with recent theoretical calculations. The nuclear quadrupole moment is derived from b to be Q(VLi) = -41(6) mbarn.

In male Sprague-Dawley rats, renal artery constriction in the presence of an inact contralateral kidney induced sodium retention (for 2-3 wk), moderate potassium loss,elevation of blood volume (BV), and an increase in water turnover. It is suggestedthat renal artery constriction activates the renin-angiotensin-aldosterone system, resulting in disordered regulation of salt and water balance and in blood pressure (BP) elevation. Subsequently, sodium balance was reestablished in one group of hypertensive rats. The previously retained sodium was kept in the body, and BV and reninactivity remained elevated. In a second group of animals, a malignant course of hypertension developed: BP surpassed a critical level of about 180 mmHg; sodium, potassium, and water were lost; BV declined; renin activity was further stimulated; and in the contralateral kidney malignant nephrosclerosis occurred. It is assumed that pressure diuresis and natriuresis induce a vicious circle: the increasing renin activity may maintain or further increase BP level, therby inducing further salt and water loss, etc.; high BP levels and high renin activities induce vascular damage and deterioration of renal function.

For the determination of creatine kinase isoenzyme catalytic concentrations in serum two methods based on immunological reactions are presented: One method uses inhibiting antibodies, which selectively block the activity of creatine kinase M subunits ("Inhibition Test*'). This test is used for routine measurements of creatine kinase MB catalytic concentration; Another method uses precipitating antibodies, which allows a quantitative differentiation of creatine kinase isoenzymes MM, MB and BB ("Precipitation Test"). This test is used as a control for the Inhibition Test for the possible presence of creatine kinase BB activities in doubtful cases. Procedures, specificity, correlation and application of these methods are discussed. Quantitative Bestimmung der katalytischen Konzentrationen von Creatinkinase-Isoenzymen im Serum mit Hilfe immunologischer Methoden Zusammenfassung: Für die Bestimmung der katalytischen Konzentrationen von Creatinkinase-Isoenzymen im Serum werden zwei Methoden auf immunologischer Basis beschrieben: Eine Methode unter Verwendung inhibierender Antikörper, welche die Aktivität der Creatinkinase-M-Untereinheiten selektiv hemmen ("Hemmtest"). Diese Methode wird zur routinemäßigen Bestimmung der katalytischen Konzentrationen von Creatinkinase-MB verwendet; Eine zweite Methode unter Verwendung präzipitierender Antikörper, welche die quantitative Differenzierung der Creatinkinäse-Isoenzyme MM, MB und BB erlaubt. Diese Methode wird in Zweifelsfällen als Kontrolle auf eventuelle Creatinkinase-BB-Aktivitäten verwendet. Die Methoden, ihre Spezifität, Korrelation und Anwendungsmöglichkeiten werden diskutiert.

Pepstatin, an N-acylated pentapeptide obtained from culture filtrates of actinomycetes and first characterized as an inhibitor of pepsin, produces inhibition of the renin-substrate reaction both in vitro and in vivo.

We report the results of a measurement of the Lamb shift in the n =2 state of muonium. Muonium atoms in the metastable 2S state were produced by the beam-foil method and the formation rate was determined by observation of the Ly-a photons emitted when the metastable atoms were quenched in a static electric field. The Lamb shift (4'&") was measured through observation of the microwave-induced 2'S&&2-2'P&&2 transition with the result $&"=1042» MHz. This result is in agreement with the current theoretical value.