Extraction and purification of molybdenum cofactor from milk xanthine oxidase.

Molybdenum cofactor (mocofactor) is extracted efficiently, free of impurities and in high concentrations, by acid treatment of xanthine oxidase and subsequent incubation of the precipitate with phosphate buffer containing EDTA, molybdate and oxygen. It is suggested that cofactor is bound to the enzyme via hydrophobic forces as well as via an oxygen-sensitive mechanism. Upon extraction, the capability to complement the apo nitrate reductase of Neurospora crassa nit-1 can be conserved only in the total absence of oxygen. Cysteine and glutathione were shown to protect efficiently free mocofactor from oxidation. Two species of active mocofactor, probably a molybdoform and a demolybdoform, could be separated by means of reversed-phase HPLC with a mobile phase of 5 mM sodium citrate at a pH of 6.5. The mode of interaction between either of these species with thiol reagents is discussed.

The oxidation product of molybdenum cofactor from milk xanthine oxidase.

In extracts of acid treated molybdenum cofactor containing xanthine oxidase, fluorescence is maximally developed upon a three hours incubation. Analysis by means of reversed phase HPLC revealed the presence of several fluorescent compounds, the main one being a blue fluorescent compound with an emission maximum of 465 nm when maximal excited at 395 nm at a neutral pH. Definite proof is presented that this compound is the oxidation product of the molybdenum cofactor. The remaining fluorescent products are shown to be pterin-derivatives, yielding predominantly pterin-6-carboxylic acid upon permanganate oxidation. Purified oxidation product of molybdenum cofactor however, didn's yield a fluorescent derivative at all upon treatment with permanganate.

Mass spectrometric analysis of fluorescent products originating from the molybdenum cofactor.

Mass spectra of 4 fluorescent HPLC fractions originating from the molybdenum cofactor in xanthine oxidase extracts have been obtained with the method of field desorption (FD). The most polar fraction contains compounds which show peaks in the M/Z = 110-230 and M/Z = 580-750 range. Two other fractions exhibit in the FD spectra peaks at M/Z = 1,113 and M/Z = 886, respectively. Both corresponding compounds contain at most 24 C atoms and lack S, Mo, Cl and Br, as judged from the isotopic pattern. The most apolar fluorescent compound, which could be isolated only from xanthine oxidase extracts prepared in the absence of phosphate, has been identified as a species with a molecular weight around 482.

Growth yields, polysaccharide production and energy conservation in chemostat cultures of Rhizobium trifolii.

Rhizobium trifolii was grown in a defined medium in chemostat cultures. Extracellular polysaccharide production was found in carbon-sufficient as well as in carbon-limited cultures. Extracellular polysaccharide production in carbon-limited cultures was strongly dependent on the growth rate. In mannitol-limited cultures, asparagine was always totally depleted from the culture medium. Only when the asparagine supply was not sufficient to meet the nitrogen need of the culture, ammonia assimilation took place. Excess organic nitrogen was excreted as ammonia. Whether ammonia assimilation or ammonia excretion took place was also dependent on the growth rate. Respiration-coupled proton translocation measurements showed the presence of three energy conserving sites in an electron transport chain which is branched. Assuming a H+/P ratio of 4, a P/O ratio of 2.33 was found. Growth yield calculations indicated a P/O ratio of approximately 2. Sulphate limitation in the chemostat culture resulted in a decrease in the efficiency of oxidative phosphorylation and in a less stringent coupling between growth and energy yielding processes.

A continuous culture study of an ATPase-negative mutant of Escherichia coli.

For anaerobic glucose-limited chemostat cultures of Escherichia coli a value of 8.5 was found for YmaxATP. For anaerobic glucose- or ammoniumlimited chemostat cultures of the ATPase-negative mutant M2-6 of E. coli YmaxATP values of 17.6 and 20.0 were found, respectively. From these data it can be concluded that in the wild type during anaerobic growth 51-58% of the total ATP production is used for energetization of the membrane. Using the YATP values obtained in the anaerobic experiments a P/O ratio of 1.46 could be calculated for aerobic experiments with the wild type. It is concluded that from the energy obtained by respiration in wild type E. coli about 60% is used for membrane energetization and only about 40% for the actual formation of ATP. No dramatic difference in the maintenance requirement for ATP or glucose has been observed between glucose- and ammonium-limited chemostat cultures of the mutant. The large difference in maintenance requirement observed for such cultures of the wild type is therefore supposed to be made possible by ATP hydrolysis by the ATPase.

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium.

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and YATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y max ATP and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth-dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a mu value of 1.44 h-1 the molar growth yield for glucose was about 70 and Y ATP about 28.5. An equation is presented, which gives the relation between theoretical and experimental Y max ATP values.

Determination of the efficiency of oxidative phosphorylation in continuous cultures of Aerobacter aerogenes.

For anaerobic glucose-limited chemostat cultures of Aerobacter aerogenes a values of 14.0 g/mole was found for Ymax/ATP and a value of 6.8 mmoles ATP/g dry weight/hr for the maintenance coefficient. Both values are much lower than those previously determined for tryptophan-limited anaerobic chemostat cultures. It is concluded that generally the largest part of the maintenance energy is not used for true maintenance processes. For aerobic glucose-limited chemostat cultures two phases could be differentiated. Acetate production started at mu values higher than 0.53. The slopes of the curves relating the specific rates of glucose- and oxygen consumption with mu became higher and lower respectively above the mu value of 0.53. Using the YATP values obtained in the anaerobic experiment a P/O ratio of about 1.3 could be calculated for glucose- and tryptophan-limited chemostat cultures. In sulfate-limited chemostat cultures acetate was produced at all growth rates. At high growth rates also pyruvate and alpha-ketoglutarate were produced. With the YATP values obtained in the anaerobic experiment a P/O ratio of about 0.4 was calculated for sulfate-limited chemostat cultures.