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...

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The effects of the thiadiazinone derivative, 5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydrochinolin-6-yl]-6-met hyl-3,6- dihydro-2H-1,3,4-thiadiazin-2-on (EMD 53998), and of its (+)EMD 57033 and (-)EMD 57439 enantiomers, were tested on the contractile properties and cytosolic [Ca2+] ([Ca2+]i) transients of single intact guinea pig cardiac myocytes. Cells were loaded with the ester form of the fluorescent probe, indo-1, and bathed in a N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid-buffered solution at 25 degrees C (1 mM of CaCl2, 1 Hz stimulation rate). All three substances exerted a pronounced increase in twitch amplitude: the maximal effect of the racemate (380% of control value) was approximately the sum of the effects of its two enantiomers (186 and 236% of control value for the (+)- and (-)-enantiomer, respectively). The [Ca2+]i transient, measured as the 410-to-490 nm indo-1 fluorescence ratio transient after excitation, was increased by the racemate and its (-)-enantiomer (172 and 152% of control value, respectively), but was not increased by the (+)-enantiomer. The racemate and the (-)-enantiomer, but not the (+)-enantiomer, markedly reduced the contraction duration and [Ca2+]i transient duration. In unstimulated cells resting length was significantly reduced by the (+)-enantiomer, and this was accompanied by a decrease in indo-1 fluorescence; the (-)-enantiomer had no effect on either parameter. In the presence of 2,3 butanedione monoxime (BDM), which markedly reduces twitch amplitude by inhibiting cross-bridge mechanics, addition of the (+)-enantiomer restored the twitch contraction to above the pre-BDM level without augmenting the [Ca2+]i transient. In contrast, the (-)-enantiomer failed to reverse the BDM-induced contractile depression, even though it caused a significant increase of the [Ca2+]i transient. Thus, in intact cells the positive inotropic effect of EMD 53998 is due to specific properties of its enantiomers: the (-)-enantiomer has adenosine 3',5'-cyclic monophosphate-like effects (increase in amplitude and reduction of [Ca2+]i transient and contraction durations), whereas the (+)-enantiomer enhances the myofilament-Ca2+ interaction.

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The inotropic state of the myocardium can be enhanced via an increase in cell Ca2+ loading or in myofilament responsiveness to Ca2+. Although different pharmacological agents combine these properties, no presently available drug acts predominantly as a myofilament sensitizer in situ. We have investigated the effects and the mechanism of action of novel diazinone derivatives, EMD 54622, EMD 53998, and EMD 54650 (developed by E. Merck, Darmstadt), on guinea pig myocardial preparations. Force- and ATPase-pCa relations in skinned fibers show differing potencies of these agents on myofilament sensitization: EMD 54622 greater than EMD 53998 much greater than EMD 54650. This is in contrast to their relative potencies to inhibit isolated myocardial phosphodiesterase III: EMD 54650 greater than EMD 53998 greater than EMD 54622. In isolated hearts studied at constant coronary flow, each of the three diazinone derivatives had a positive inotropic effect. In enzymatically dissociated left ventricular myocytes loaded with the Ca2+ probe indo-1, the positive inotropic effect of EMD 54622 occurred with no change in the amplitude of the cytosolic [Ca2+] (Cai) transient. In contrast, both EMD 53998 and EMD 54650 enhanced Cai transient and twitch contraction amplitudes. Length-indo-1 fluorescence relations were analyzed to determine the effects of the three substances on myofilament responsiveness to Ca2+. EMD 54622 enhanced and EMD 54650 had no effect on myofilament responsiveness to Ca2+. Less uniform results were obtained with EMD 53998 (in two of five cells the myofilament responsiveness to Ca2+ was increased, whereas in three other cells it was unaltered). Our results indicate that structural changes in the diazinone molecule shift the mechanism of action for the positive inotropic effect of the diazinone derivatives in the intact cell from a predominant myofilament sensitization (EMD 54622) to an enhancement in cell Ca2+ loading and an augmentation in the Cai transient (EMD 54650).

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