A strain of Pseudomonus putida grown on 4-methoxybenzoate as sole carbon source contains an enzyme system for the 0-demethylation of this substrate. The enzyme system is purifiable and can be separated into two components : an NADH-dependent reductase and an iron-containing and acid-labile-sulfur-containing monooxygenase.The reductase, of molecular weight 42000 and containing two chromophores, an FMN and an iron-sulfur complex (EPR at g = 1.95), reduces both one-electron and two-electron acceptors (i.e., ferricyanide, 2,6-dichloroindophenol, cytochrome c, and cytochrome b5) at an optimum pH of 8.0. Increasing ionic strength affects these activities differently.The absolute spectrum of the oxidized reductase displays distinct absorption peaks at 409 and 463 nm and a small shoulder between 538 and 554 nm. Treatment with dithionite or NADH reduces the absorbance throughout the visible range, yielding a spectrum with small maxima at 402 and 538 nm. Spectroscopic characteristics of the reductase indicate a tight coupling between its two chromophores.The iron-containing and acid-labile-sulfur-containing monooxygenase, which has a molecular weight of about 120000, contains an iron-sulfur chromophore with an EPR signal at g = 1.90. This protein is a dimer whose subunits each have a molecular weight of about 50000 and are perhaps identical. The optical absorption properties are somewhat unusual. In contrast to other iron-sulfur proteins, there is no significant peak near 41 5 nm in the absorption spectrum of the oxidized protein, but rather one at 455 nm. The presence of the substrate 4-methoxybenzoate increases both the velocity and the extent of reduction of the iron and labile-sulfur-containing monooxygenase by the NADH-dependent reductase.Hydroxylation can be achieved by the monooxygenase also in absence of the reductase with artifical reductants. This enzyme opens a new group of oxygenases within the classification scheme, i e . , iron-containing and labile-sulfur-containing monooxygenases.From the reported data, a scheme for the interaction of the isolated pigments and their relationship to various acceptors is proposed. The 4-methoxybenzoate 0-demethylase from Pseudomonas putidu is a fairly unspecific enzyme. In the presence of NADH and molecular oxygen, the aliphatic C-H bond as well as the aromatic ring are attacked [ll]. Studies on substrate binding and oxygen aromatic ring, rather than the side chains of the Abbreviations. CD, circular dichroism; EPR, electron paramagnetic resonance, 2, mean g-value = 1/3 (gx + g, + gz).
Phospholipid metabolism has been investigated in embryonic rat fibroblasts which have been incubated in serum-free medium and subsequently stimulated by the growth-stimulating serum proteins S1 and S2:.1. Incorporation of 32P into phosphatidylinositol increases rapidly within a few minutes of stimulation; after 30 min it is 7.4-fold higher in stimulated cells when compared with the controls. Phosphatidylcholine synthesis and uptake of 32P into the acid-soluble pool is not significantly enhanced during this time. Labelling of phosphatidylserine, phosphatidylethanolamine and sphingomyelin during this period is just above the background level.2. Incorporation of 32P into phosphatidylinositol and phosphatidylethanolamine is strongly inhibited in the presence of dibutyryl-adenosine 3' : 5'-monophosphate and theophylline whereas the metabolism of phosphatidylcholine is not influenced.As demonstrated with [jH]inositol, the inhibition of phosphatidylinositol synthesis is restricted to dibutyryladenosine 3' : 5'-monophosphate; adenosine 3' : 5'-monophosphate; guanosine 3' : 5'-monophosphate and dibutyryl guanosine 3' : 5'-monophosphate are ineffective.3 . The growth-stimulating serum factors must be present continuously for activation of phosphatidylinositol synthesis; within 1 h after serum deprivation the incorporation of 32P into phosphatidylinositol decreases to about 20 of the original value ; in contrast, phosphatidylcholine biosynthesis is not influenced significantly during this time period.
The phospholipids of embryonic rat fibroblasts in tissue culture have been labelled with [3H] (myo) inositol, [3H] choline, [3H] glycerol and inorganic 32P , respectively. After 24 hours in serum-free medium the cells have been triggered by fetal calf serum or by a mixture of the growth-stimulating serum proteins S1 and S2, and the phospholipids were examined.
1. Up to 15 - 20 percent of total radioactivity are eliminated from the phospholipid fraction during 30 min after stimulation, when the cells have been prelabelled with [3H ]inositol; within 2 hours, however, the original value is restored. When the cells are prelabelled with 32P, the radio activity also decreases, but in contrast to the experiments with [3H] inositol it remains low. From this result one must conclude that part of the PI in the membranes is broken down after stimula tion, and that the inosit moity is reutilized for newly synthesized PI. After stimulation, a small amount of labelled PI is also found in the culture medium .
2. The elimination of radioactivity does not exclusively take place in phase. It can also be demonstrated with cells in S after removal of serum for one hour and readdition.
3. The loss of radioactivity cannot be demonstrated when the cells are triggered by dialysed fetal calf serum or by the serum factors S1 and S2. Examination of the dialysate revealed phosphatidvlinositol as compound which necessarily must be present for the elimination process after stimulation. This elimination process is also demonstrable if the biosynthesis of PI is blocked by gamma-or delta-hexachlorocyclohexane, whereas the beta derivative is completely ineffective.
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