Manfred Kempfle

Composition, N-terminal amino acids, and chain length of a β-glucosidase from Aspergillus wenth

Abstract 1. 1.|The β-glucosidase A 3 (β- D -glucoside glucohydrolase, EC 3.2.1.21) from Aspergillus wentii is an acidic glycoprotein of molecular weight 170 000 (G. LEGLER , Z. Physiol. Chem. , 348 (1967) 1359). Its isoelectric point was pH 3.80. Except for the preponderance of acidic amino acids and the presence of only 4 disulfide bonds, no unusual features were found in the amino acid composition. The protein contained about 21% carbohydrate, including 6.2% glucosamine. Mannose, and smaller amounts of glucose and fucose were identified as neutral sugars. 2. 2.|Determination of the N-terminal amino acids by the dansyl method gave 1 mole aspartic acid (or asparagine) per 170 000 g protein. 3. 3.|Attempts to dissociate the enzyme into subunits by reduction of the disulfide bonds, subsequent reaction with 5,5′-dithiobis-(2-nitrobenzoic acid), acrylonitrile, or ethyleneimine, and chromatography or electrophoresis in the presence of denaturing agents were unsuccessful. 4. 4.|The intrinsic viscosity of the reduced and denatured enzyme indicated a chain length between 910 and 1300 amino acid residues. 1200 residues are calculated from the amino acid analysis. 5. 5.|The molecular weight, determined by sedimentation analysis of the reduced and alkylated enzyme in 6 M guanidine hydrochloride, was 168 000 ± 5000. It is concluded that the enzyme consists of a single peptide chain of about 1200 amino acid residues. 6. 6.|ORD and CD measurements indicated that only about 15% of the molecule was present in a helical conformation. Conformational changes set in at 20° below pH 2.2 and above pH 7.5.

The binding of fluorescent 4,6,8(14)-triene-3-one steroids to cyclodextrins as a model for steroid—protein interactions

The 4,6,8(14)-triene-3-one steroids, highly fluorescent in aqueous solutions, lose their fluorescence power when binding occurs to hydrophobic regions of other molecules, such as the hydrophobic cavity in the ring system of cyclodextrins. The fluorescence intensity decreases almost completely when beta- and gamma-cyclodextrins are present in the solution. Scatchard plots derived from fluorescence titrations show that one or two molecules of steroid bind to one cyclodextrin molecule with KD,F-values of about 10(-4)-10(-5) mol/liter. Temperature-jump experiments show a single relaxation process, with rate constants for the decay of the beta-cyclodextrin-steroid complexes of about 10(4)-10(5) per s. For alpha- and gamma-cyclodextrins such relaxation processes are not observed.

Characterization of the hydrophobic interaction of steroids with endoplasmic reticulum membranes by quenching of 6,8(14)-bisdehydro-17α-hydroxyprogesterone fluorescence

The fluorescence behaviour of 4,6,8(14)-trien-3-one steroids, which exhibit fluorescence in protic media but no fluorescence in hydrophobic environments, was used to characterize the molecular nature and temperature-sensitivity of steroid hormone-biomembrane interactions. Since 17 alpha-hydroxyprogesterone as the key intermediate is known to accumulate in smooth endoplasmic reticulum membranes in the course of adrenal and testicular steroid hormone biosynthesis, its fluorescent analogue, 6,8(14)-bis-dehydro-17 alpha-hydroxyprogesterone (BDHP), was used as the probe molecule. With rat testis microsomal membranes and liposomes, fluorescence quenching in the presence of membranes (related to fluorescence in aqueous solution) was independent on steroid concentration but was dependent on membrane lipid concentration in terms of a hyperbolic function. Complete fluorescence loss occurred at infinite lipid concentration at 20 degrees C, indicating complete insertion of the steroid probe into the hydrophobic portion of the membrane compartment. The partition coefficient KP increased with increasing temperature as a consequence of increased membrane fluidity. The result that BDHP fluorescence decreased considerably with elevated temperature in both the aqueous and the membrane milieu was interpreted as the consequence of increasing molecular mobility; this effect was much more pronounced in the aqueous than in the membrane environment. On the basis of local BDHP concentrations within the membrane phase (calculated from KP), relative fluorescence quenching was over-proportional at low temperatures; under that condition, hydrophobic interactions with rigid membrane lipid domains are obviously favoured.

Fluorescence of 3-keto-steroids in aqueous solution

The physiologically important 3-keto-steroids are non-fluorescent or only weakly fluorescent in protic as well as in aprotic solvents. In contrast, the 4,6,8(14)-triene-3-one steroids are highly fluorescent in aqueous solution but they do not appreciably fluoresce in other solvents. Evidence is presented that the introduction of double bonds into the skeleton of the 3-keto-steroids leads to a decrease of the energy of the lowest π − π* state, bringing this level into the neighbourhood of the non-fluorescent n − π* state. As a consequence, for two states of approximately the same energy, relatively small perturbations such as those due to solvent interactions, protein binding and micelle formation, will then determine whether a system will fluoresce (π − π* state lowest) or not (n − π* state lowest). When the fluorescent 3-keto-steroids, having three conjugated double bonds, bind to proteins, the fluorescence intensity becomes almost zero, making these compounds useful as probes for steroid-protein interactions. This quenching of the fluorescence is explained by a decrease in energy of the n − π* state relative to the π − π* state of the steroids due to hydrophobic interactions with the proteins.

Relaxation kinetic studies of coenzyme binding to glutamate dehydrogenase from beef liver

Fluorescence temperature-jump experiments were performed to study the binding of coenzyme to glutamate dehydrogenase from beef liver in 0.1 M sodium phosphate buffer, pH 7.4, T = 25.0°C. 1. 1) NADPH shows a single second-order relaxation process, indicating coenzyme binding to one site per enzyme subunit. The dissociation constant of the reaction was calculated from the rate constants (k21 = 92 sec−1, k12 = 2.13 .106 M−1 sec−1) to be 43 μM. No deviation from a straight line was observed over the whole concentration range. 2. 2) With NADH two well separated second-order processes are detected. 3. 3) The observed concentration dependence of these two relaxation processes is consistent with a reaction scheme assuming two completely independent but non-equivalent NADH binding sites per enzyme subunit. 4. 4) From the rate constants of the faster process (k21 = 235 sec−1 k12 = 3.83 .106 M−1 sec−1) and of the slower process (k32 = 4.2 sec−1, k23 = 0.18 .106 M−1 sec−1) the dissociation constants are calculated to be K21 = k21k12 = 61 μM and K32 = k32k23 = 23 μM, respectively. 5. 5) Small amounts of ADP (<10 μM) abolish the slower relaxation process, while the faster (but weaker binding) process remains uninfluenced. Therefore, the weaker binding site must be the active site.

Bestimmung von Enzym-Coenzym-Dissoziationskonstanten durch Verdrängung von Tetrajodfluorescein aus den Enzym-Farbstoff-Komplexen

Tetraiodofluorescein (Erythrosin B) is bound to several dehydrogenases. This binding becomes measureable by an absorption difference spectrum in the wavelength range between 500 nm and 600 nm. Apparently the dye is bound to the adenosine moiety of the active site of nicotineamide dependent dehydrogenases. Thus, displacement of the dye should occur by several coenzymes and nucleotides and hence gives valuable information about the binding properties of these sites.The dissociation constant of enzyme-coenzyme complexes is given byKD,EL=K · KD,EF whereKD,EF is the dissociation constant of the enzyme-dye complex andK is the equilibrium constant between dye and coenzyme during the displacements.The method described is used to determine the dissociation constants of NAD+ and NADH on alcoholdehydrogenase from yeast and horse liver and on lactate dehydrogenase from beef and rabbit sceletal muscle. The results are compared with known values.ZusammenfassungDie Bindung des Farbstoffs Tetrajodfluorescein an verschiedene Dehydrogenasen wird herangezogen, um die Dissoziationskonstanten von Enzym-Coenzym-Komplexen zu bestimmen, da der Farbstoff und pyridinhaltige Coenzyme um die gleiche Bindungsstelle konkurrieren. Durch Zugabe der interessierenden Coenzyme läßt sich somit der Farbstoff aus seiner Bindungsstelle am Enzym verdrängen.Die Bindung und die Verdrängung des Farbstoffs läßt sich durch Differenzspektrophotometrie untersuchen, wobei die Eigenschaften der Enzym-Farbstoff- und der Enzym-Coenzym-Komplexe aus den Differenzspektren entnehmbar sind. Dabei ergibt sich folgender Zusammenhang:KD,EL=K · KD,EF, d.h. die gesuchte Dissoziationskonstante des Enzym-Coenzym-Komplexes ist das Produkt aus der Dissoziationskonstanten des Enzym-Farbstoff-Komplexes und der „Kopplungskonstanten“K zwischen Farbstoff und Coenzym bei der Verdrängungsreaktion.Um Anwendbarkeit und Genauigkeit dieser Methodik zu zeigen, werden die Dissoziationskonstanten von NAD+ bzw. NADH an Alkoholdehydrogenase aus Hefe und Pferdeleber und an Lactatdehydrogenase aus Rinder- und Kaninchenmuskel bestimmt und mit den bekannten Werten verglichen.

Molekulargewichtsbestimmung im Mikrogrammbereich mit Hilfe der analytischen Ultrazentrifuge

The ultraviolet absorption of the molecules is utilized, but simultaneously a caesium chloride density gradient is employed. This causes an aggregation of the substance in a very small range of the cell. Thus it is possible to work with quantities far below those needed with other methods (1 to 10 μg/ml of solution).Registration with a photomultiplier and scanning of the absorption distribution by a recorder gives a gain of time in contrast to a photographic exposure and a considerable improvement in accuracy.The molecular weight range is limited between 10,000 and 20 millions by the susceptibility time of the recorder.ZusammenfassungEine Methode wird beschrieben, die eine Bestimmung von Molekulargewichten im Mikrogrammbereich mit Hilfe einer analytischen Ultrazentrifuge ermöglicht. Dabei wird die Ultraviolettabsorption der Substanzen ausgenutzt, gleichzeitig aber ein Caesiumchlorid-Dichtegradient angewendet, durch den eine Ansammlung der Substanz in einem schmalen Bereich der Zelle erzwungen wird. Dies ermöglicht ein Arbeiten mit Mengen, die weit unter denen liegen, die bei anderen Methoden benötigt werden (etwa 1–10 μg/ml Lösung, der Bedarf an Lösung beträgt etwa 0,5 ml).Durch Registrierung mit einem Sekundärelektronenvervielfacher und unmittelbarer Aufzeichnung des Absorptionsverlaufs durch einen Schreiber wird gegenüber einer Registrierung mit einer Photoplatte ein Zeitgewinn erzielt und die Genauigkeit beträchtlich verbessert.Infolge der Ansprechempfindlichkeit des Schreibers ist die Anwendung dieser Methode jedoch auf einen Molekulargewichtsbereich von etwa 10000 bis zu etwa 20 Millionen beschränkt.

Analytische Dichtegradienten-Zentrifugation bei Temperaturen zwischen +4‡C und +40‡C

It is shown that all the parameters needed for analytical density gradient centrifugation tabulated by Hearst et al. for 25‡C are valid for other temperatures, too. This will be important in the investigation of highly sensitive enzymes which are only stable at temperatures near +4‡C over a period of 24 h. Comparison of molecular weights determined at 6‡C und 25‡C shows no difference greater than 2%, which will be within the experimental error.ZusammenfassungEs wird gezeigt, da\ die bei der analytischen Dichtegradienten-Zentrifugation benötigten Parameter, die von Hearst u. Mitarb. tabelliert wurden, auch für andere Temperaturen als 25‡C gültig sind. Das ist von gro\er Wichtigkeit, da hochempfindliche Enzyme nur bei Temperaturen um + 4‡C lÄnger als 24 h nativ bleiben. Man ist deshalb auf eine Zentrifugation bei dieser Temperatur angewiesen. Weil man oft nur sehr wenig Substanz isolieren kann, bleibt nur die Dichtegradienten-Zentrifugation als Methode der Wahl. Ein Vergleich der gemessenen Molekulargewichte bei 6‡C und 25‡C zeigt keine Abweichungen, die über die Me\genauigkeit hinausgehen.