Cytochrome b561 spectral changes associated with electron transfer in chromaffin-vesicle ghosts

The involvement of cytochrome b561, an integral membrane protein, in electron transfer across chromaffin-vesicle membranes is confirmed by changes in its redox state observed as changes in the absorption spectrum occurring during electron transfer. In ascorbate-loaded chromaffin-vesicle ghosts, cytochrome b561 is nearly completely reduced and exhibits an absorption maximum at 561 nm. When ferricyanide is added to a suspension of these ghosts, the cytochrome becomes oxidized as indicated by the disappearance of the 561 nm absorption. If a small amount of ferricyanide is added, it becomes completely reduced by electron transfer from intravesicular ascorbate. When this happens, cytochrome b561 returns to its reduced state. If an excess of ferricyanide is added, the intravesicular ascorbate becomes exhausted and the cytochrome b561 remains oxidized. The spectrum of these absorbance changes correlates with the difference spectrum (reduced-oxidized) of cytochrome b561. Cytochrome b561 becomes transiently oxidized when ascorbate oxidase is added to a suspension of ascorbate-loaded ghosts. Since dehydroascorbate does not oxidize cytochrome b561, it is likely that oxidation is caused by semidehydroascorbate generated by ascorbate oxidase acting on free ascorbate. This suggests that cytochrome b561 can reduce semidehydroascorbate and supports the hypothesis that the function of cytochrome b561 in vivo is to transfer electrons into chromaffin vesicles to reduce internal semidehydroascorbate to ascorbate.     

Lysine biosynthesis inMethanobacterium thermoautotrophicum is by the diaminopimelic acid pathway

Methanobacterium thermoautotrophicum, an archaebacterium, possesses the first and last enzymes of the diaminopimelic acid pathway for lysine biosynthesis, dihydrodipicolinate synthase, and diaminopimelate decarboxylase. It does not have saccharopine dehydrogenase, the last enzyme of the aminoadipate pathway for lysine biosynthesis. The dihydrodipicolinate synthase is inhibited but not repressed by lysine. We conclude that this microbe uses the diaminopimelate pathway for synthesis of lysine.Deceased.     

Improved Stability of Methanolic Wright's Stain with Additive Reagents

Additive reagents have been investigated to improve the stability of methanolic Wright's stain. The addition of ammonium halides, monoalkyiamine hydrochlorides, dialkylamine hydrochlorides or trialkylamine hydrochlorides to methanolic Wright's stain was found to enhance the stability of stain components in methanol. No change in performance is observed with these additives present. Random precipitation in the stain solution was still observed with the addition of ammonium halides and monoalkyiamine hydrochlorides. No precipitation was found in stain solutions containing hydrochlorides of most dialkylamines and trialkylamines. Of the compounds evaluated, 0.6% diethylamine hydrochloride added to methanolic stain solutions produced the most desirable overall results. Mechanisms of stabilization and precipitation in these stain solutions are proposed, Essentially, separation of the thiazine-eosinate ion pair through interaction with an appropriate additive increases stain stability. The solubilities of thiazine-eosinate or additive cation-eosinate ion pairs in methanol determine the formation of precipitate in such stain solutions.