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.

---

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.

     

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.     

Enzyme clusters during the metamorphic period of Ambystoma mexicanum: role of thyroid hormone

Enzyme activities and DNA content have been measure in axolotl liver during the metamorphic period (4-8 months after spawning). Three different types of enzyme activity profiles were observed. In the type I profile (carbamoyl-phosphate synthase, arginase, ornithine transcarbamoylase, and glutamate dehydrogenase) enzyme activity is high in the youngest animals studied, and shows a minimum at 5 months followed by a maximum at 8 months of age. Thereafter activities do not change or slightly decrease. In the type II profile (tyrosine aminotransferase, glucose-6-phosphatase) enzyme activity shows a peak at 5 months of age and is low thereafter. Hexokinase, the enzyme with a type III profile, shows high activity throughout the metamorphic period. DNA content remains high throughout the metamorphic period but decreases 50% between 9 and 12 months of age, probably due to an increase in the size of the hepatocytes. No glucokinase activity was detected. High activities of cluster II enzymes represent early metamorphic events, while the rising part of cluster I is associated with late metamorphic events. The apparent molecular specific activity increases during natural development between 5 and 9 months of age, or precociously, upon thyroid hormone treatment. This change in apparent molecular specific activity is correlated to the advent of ureotelism.     

The catalytic mechanism of cytochrome P-450. Spin-trapping evidence for one-electron substrate oxidation

Cytochrome P-450 is destroyed during catalytic oxidation of several 4-substituted 3,5-bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine substrates. A qualitative correlation has been found between the ability to destroy cytochrome P-450 and the stability of the 4-substituent as a radical. Destruction of the enzyme by the 4-ethyl (DDEP), 4-propyl, and 4-isobutyl analogues is due to transfer of the 4-alkyl group from the substrate to a nitrogen of the prosthetic heme, a process which gives rise to isolable N-alkylprotoporphyrin IX derivatives. Little enzyme destruction is observed when the 4-alkyl group is of low radical stability (methyl, phenyl) and good destruction, but no isolable heme adducts when the 4-substituent is of very high radical stability (isopropyl, benzyl). Spin-trapping studies have established that the 4-ethyl group in DDEP is lost as a radical as a result of oxidation by cytochrome P-450. Of three commonly used spin traps, only alpha-(4-pyridyl-1-oxide) N-tert-butylnitrone was found suitable for such studies. The other spin traps, 5,5-dimethyl-1-pyrroline-N-oxide and alpha-phenyl N-tert-butylnitrone, were found to be ineffective, the latter because it strongly inhibits cytochrome P-450. Hydrogen peroxide formed in situ can support a part of the cytochrome P-450-catalyzed ethyl radical formation and DDEP-dependent self-inactivation. The results provide persuasive evidence that oxidation of the nitrogen in DDEP by cytochrome P-450 proceeds in one-electron steps. Cytochrome P-450 may thus function, at least with certain substrates, as a one-electron oxidant.     

Auxin controls Golgi-localized glucan synthetase activity in the maize mesocotyl

Decapitation or red light irradiation (R) inhibited growth and Golgi-localized glucan synthetase (GS I) activity in the mesocotyl of intact maize (Zea mays L.) seedlings. Applied auxin (indole-3-acetic acid) prevented the effects of R and of decapitation on both growth and GS I. Auxin applied several hours after irradiation prevented any further decline in GS I but did not restore it. Mesocotyl segments incubated in solution elongated in response to auxin but lost GS I with time regardless of the presence of exogenous auxin. An attached seed was necessary for maintenance of GS I in the dark-grown mesocotyl.Abbreviations GS glucan synthetase - IAA indole-3-acetic acid - R red light     

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.