The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.     

Quantitative Bestimmung von Sulfhydrylgruppen mit Mercurochrom

Zusammenfassung Native Ehrlich-Ascites Tumorzellen (EATZ) werden in einer 1×10^–3 M Lösung von Dibrommercurifluoreszein (DBMF) in Sörensen-Phosphatpuffer pH 6.7+0,9% NaCl 30 min inkubiert und sodann viermal mit dem gleichen Puffer, der 0,01 M in bezug auf NaCN ist, bis zur Farblosigkeit des Überstandes gewaschen. Die Zellen zeigen nun ein Absorptionsmaximum zwischen 520 und 525 nm, das mit dem von Komplexen reiner Korpuskularproteine mit DBMF identisch ist. Die Zellen werden im Scanning bei 520 nm photometriert und daraus ihre Gesamtextinktionen ermittelt. Unter Zugrundelegung des in früheren Arbeiten bei den Protein-DBMF-Komplexen bestimmten Extinktions-koeffizienten =33 000 ergibt sich ein Gehalt von rund 1,1×10^–14 Molen Proteinthiolen (Prot-SH) pro Zelle. Dieser Wert entspricht sowohl dem von Nöhammer 1982 mit Dihydroxydinaphthyldisulfid gefundenen Gehalt an reaktiven, d.h. schnell reagierenden Prot-SH, als auch dem von Rindler et al. 1970 bestimmten SH-Gehalt der primär löslichen Zellproteine. Da aus nativ mit DBMF behandelten Zellen jedoch keine im Homogenat-Puffer löslichen Proteine gefunden wurden, ist es wahrscheinlich, daß DBMF mit den schnellen SH-Gruppen der Cytosol-Proteine reagiert, die dabei strukturelle Veränderungen erfahren, die zum Löslichkeitsverlust führen. DBMF erfaßt jedoch die gesamten Prot-SH, reaktive und maskierte, wenn es auf vorher fixierte Zellen einwirkt (Nöhammer et al. 1981). Ist der zum Waschen verwendete Puffer Cyanid-frei, so wird die identische Absorptionsbande gemessen; die für E _tot,520 ermittelten Werte sind jedoch um 60% höher. Die Differenz wird nicht-kovalent, reversibel an die Zellen gebundenem DBMF zugeschrieben.

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Quantitative determination of sulfhydryl groups with MercurochromeII. Detection of fast reacting protein thiols in native cells

Summary Native Ehrlich ascites tumor cells (EATC) are incubated during 30 min in a 1×10^–3 M solution of dibrommercurifluoresceine (DBMF) in Sörensen phosphate buffer pH 6.7+0.9% NaCl. Subsequently the cells are washed four times in the same buffer containing additionally 0.01 M NaCN until the supernate appears to be colourless. They show an absorption maximum between 520 and 525 nm which is identical with that of complexes between pure corpuscular proteins and DBMF, investigated previously. Scanning at 520 nm yields the total extinction of the stained cells E _tot which is calculated into moles of protein bound thiol groups (prot-SH), with an extinction coefficient =33,000 previously determined with the protein-DBMF-complexes. A mean protein-SH content of 1.1×10^–14 moles per single cell is found which corresponds with both the content of fast reacting prot-SH previously determined by Nöhammer with dihydroxydinaphthyldisulfide, and the SH-content of the soluble cellular proteins determined by Rindler et al. with DTNB. As no soluble proteins could be obtained from DBMF-treated cells, it can be assumed that in native cells DBMF reacts preferentially with the fast reacting SH-groups of the soluble proteins which, in the course of structural changes, became insoluble. According to Nöhammer et al. (1981), however, DBMF also reacts with the total of protein-SH content of 1.1×10^–14 moles per single cell is found which fixed with ethanol/ether. When the buffer used for washing is free from CN^–, the identical absorption band is measured; however, the values determined for E _tot,520 are approximately 60% higher. The extinction difference is ascribed to DBMF, noncovalently and reversibly bound to the cells.

     

Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

Summary 2,2-dihydroxy-6,6-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.Dedicated to Prof. Dr. E. Ziegler on the occasion of his 70^th birthday     

Determination and Health-Risk Evaluation of Nitroxynil Residues in the Edible Tissue of Cattle

A specific Polarographic method with a sensitivity of ≥ 2 jig/kg (ppb) has been used to determine the plasma and tissue concentrations of nitroxynil (NTX), which is used against Parafilaria bovicola in cattle. After treatment with the therapeutic dose on NTX (2 × 20 mg/kg b.w., s,c), there was an initial rapid decrease in the plasma concentration followed by a slower elimination phase. The plasma levels of NTX were 8 mg/kg (ppm) and 3 mg/kg after 6 weeks and 2 months, respectively. The muscle and other edible tissue from treated cattle contained 0.1–0.3 mg/kg NTX after 2 months, and jxg/kg amounts were still detectable 3 months after the injection. Based on available pharmacological and toxicological data, a 3-months withdrawal time for NTX in cattle is proposed.     

Parturient Paresis in the Cow

Serum ionized calcium concentrations (CaF) were determined in 87 Swedish red-and-white cows and 10 Swedish Friesian cows with clinical signs of parturient paresis. All cows were in the week prior to or after parturition. A classification of the severity of hypocalcemia in terms of serum ionized calcium was devised. Eight cows had normal serum ionized calcium concentrations (Cap 1.06–1.26 mmol/1); 15 had slight (CaF 0.80–1.05 mmol/1); 43 a moderate (CaF 0.50–0.79 mmol/1), and 31 asevere (CaF < 0.50 mmol/1) hypocalcemia. All cows were given 8 or 8.3 g of calcium intravenously. Of 8 normocalcemic cows 7 (87.5 %) reached a maximum posttreatment serum ionized calcium concentration > 1.80 mmol/1 (severe hypercalcemia). This was also found in 13 of 15 (86.7 %) slightly hypocalcemic cows and in 31 of 43 (72.1 %) moderately hypocalcemic cows. In the severe hypocalcemia group 14 of 31 (45.2 %) had maximum posttreatment Cap > 1.80 mmol/1). These findings emphazise the need of a rapid pretreatment evaluation of the degree of hypocalcemia. The present study also underlined the difficulty in predicting serum ionized calcium from serum total calcium concentrations.     

Replication of bacteriophage lambda DNA. Examination of variants containing double origins and observation of a bias in directionality

Bacteriophage λ variants have been constructed that possess two λ ori sites. Replicative intermediates resulting from infection with these phages have been investigated. We find that initiation of replication from the ori site on an EcoRI fragment (containing all the DNA sequences from within the red gene to the middle of gene O) cloned in the inverted orientation is predominantly bidirectional but occurs at a decreased frequency. Double initiations were observed at low frequency. However, a second cloned ori fragment (carrying two large deletions and a small insertion) cloned in the normal orientation demonstrated insignificant levels of replication from the cloned site unless the normal ori had already initiated.A bias in directionality of λ replication has been observed. Molecules that replicate unidirectionally propagate to the right more often than to the left. If the cloned ori-containing EcoRI fragment is inserted with reversed polarity, then the bias is towards the left. Bidirectional λ replicative intermediates also appear to show a similar bias but this is superimposed on a large, apparently random, effect that results in asymmetric growing-point propagation.     

The effect of electrical gradients on current fluctuations and impedance recorded fromNecturus gallbladder

Summary Transepithelial current fluctuations were recorded inNecturus gallbladder, clamped at negative as well as positive potentials up to 64 mV. With NaCl-Ringer's (+10mm TAP) on both sides a mucosa-negative potential enhanced the relaxation noise component, present at zero potential, and produced peaking in the power spectrum at potentials above –36mV. Concomitantly at these potentials an inductive as well as a capacitive low-frequency feature appeared in the impedance locus. Clamping at positive potentials of 18 mV suppressed the relaxation noise component. At potentials above 51mV the spectral values increased predominantly at low frequencies. In this case the power spectrum showed only a 1/f noise component. The experiments confirm the previous finding that a K⁺ efflux through fluctuating apical K⁺ channels exists under normal conditions. With serosal KCl-Ringer's the initial Lorentzian component was enhanced at negative but suppressed at positive potentials. The increase at negative potentials was less pronounced than in experiments with NaCl-Ringer's on both sides, indicating saturation of the fluctuating K⁺ current component. With mucosal KCl-Ringer's a negative potential depressed the initial relaxation noise component, whereas it was enhanced at +18 mV clamp potential. In the latter case an additional Lorentzian component became apparent at higher frequencies. At potentials of 36 mV and above the low-frequency Lorentzian disappeared whereas the corner frequency of the high-frequency component increased. The latter experiments demonstrate that the relaxation noise component inNecturus gallbladder consists of two superimposed Lorentzians. As the relaxation times of these two components behave differently under an electrical field, there may exist two different types of K⁺ channels. It is demonstrated that peaking in the plateau of power spectra can be explained by frequency-dependent attenuation effects, caused by a polarization impedance.