The soluble methane mono-oxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n-alkanes, n-alkenes, ethers, and alicyclic, aromatic and heterocyclic compounds.

1. Methane mono-oxygenase of Methylococcus capsulatus (Bath) catalyses the oxidation of various substituted methane derivatives including methanol. 2. It is a very non-specific oxygenase and, in some of its catalytic properties, apparently resembles the analogous enzyme from Methylomonas methanica but differs from those found in Methylosinus trichosporium and Methylomonas albus. 3. CO is oxidized to CO2. 4. C1-C8 n-alkanes are hydroxylated, yielding mixtures of the corresponding 1- and 2-alcohols; no 3- or 4-alcohols are formed. 5. Terminal alkenes yield the corresponding 1,2-epoxides. cis- or trans-but-2-ene are each oxidized to a mixture of 2,3-epoxybutane and but-2-en-1-ol with retention of the cis or trans configuration in both products; 2-butanone is also formed from cis-but-2-ene only. 6. Dimethyl ether is oxidized. Diethyl ether undergoes sub-terminal oxidation, yielding ethanol and ethanal in equimolar amounts. 7. Methane mono-oxygenase also hydroxylates cyclic alkanes and aromatic compounds. However, styrene yields only styrene epoxide and pyridine yields only pyridine N-oxide. 8. Of those compounds tested, only NADPH can replace NADH as electron donor.     

The molybdenum centre of native xanthine oxidase. Evidence for proton transfer from substrates to the centre and for existence of an anion-binding site.

The observation by Bray & Knowles [Proc. R. Soc. London Ser. A (1968) 302, 351--353] of direct transfer, during the catalytic reaction, of hydrogen atoms from substrate molecules to the enzyme xanthine oxidase was reinvestigated. The experimental phenomenon and its basic interpretation were confirmed and extended. In the reduced functional enzyme, molybdenum(V) interacts with two enzyme-bound protons, which are exchangeable with solvent protons. One of these is coupled to the metal with AHav. 1.4mT and the other with AHav. 0.3mT. The molecule also contains a site for the binding of anions, presumably as ligands of molybdenum. This is shown by effects of nitrate ions on the e.p.r. spectra. The spectra of the nitrate and 1-methylxanthine complexes of the reduced enzyme are very similar to one another, and are designated Rapid type-1 spectra. It is concluded that, in the Michaelis complex, the substrate molecule occupies the anion site, probably being bound to molybdenum via the nitrogen in its 9-position. During the turnover process, hydrogen from the substrate C-8 position, after transfer to the enzyme, appears as the proton more strongly coupled to molybdenum. This proton then exchanges with solvent deuterium with a rate constant of 27s-1, at pH 8.2 and 12 degrees C. It has been confirmed that substrate molecules occupying the anion site do not interfere with observation of the transfer and exchange processes.     

Comparison of the molybdenum centres of native and desulpho xanthine oxidase. The nature of the cyanide-labile sulphur atom and the nature of the proton-accepting group.

The non-functional form of xanthine oxidase known as the desulpho enzyme was compared with the functional enzyme in various ways, to obtain information on the structure of the molybdenum centre and the mechanism of the catalytic reaction. The desulpho enzyme, like the functional one, possesses a site for the binding of anions, presumably as ligands of molybdenum. Evidence is presented that in the Mo(V) e.p.r. signal from the desulpho-enzyme, as in that from the functional enzyme, a weakly coupled proton, in addition to a strongly coupled proton, interacts with the metal. Measurements were carried out by e.p.r. on the rate at which the proton strongly coupled to molybdenum exchanged, on diluting enzyme samples with 2H2O. For the desulpho enzyme the exchange rate constant was 0.40s-1, at pH 8.2 and 12 degrees C, and for the functional enzyme it was 85 s-1. It is shown that the great majority of reported differences between the enzyme forms are consistent with functional enzyme containing an (Enzyme)-Mo=S grouping, replaced in the desulpho form by (Enzyme)-Mo=O. Protonation of these groups, with pK values of about 8 and 10 respectively, would give (Enzyme)-Mo-SH and (Enzyme)-Mo-OH, these being the forms observed by e.p.r. The accepting group in the functional enzyme, for the proton transferred from the substrate while molybdenum is reduced in the catalytic reaction [Gutteridge, Tanner & Bray (1978) Biochem J. 175 869-878], is thus taken to be Mo=S.     

A fermentation process for producing both ethanol and lysine-enriched yeast.

In 18 batch-fermentation experiments, baker's yeast was grown in an enriched mineral medium, containing 10% by weight glucose, at various pH and temperature levels. The pH and temperature are just two representative engineering variables which can be easily varied at negligible cost. The commercial yeast inoculum, 20% by weight or about .16% viable cells, was selected to represent industrial (nonsterile) conditions. Free L-lysine, ethanol, and cell growth were followed in time for each batch run held at a fixed pH and temperature. The maximum free lysine level reached at either 10 1/2 or 24 hr occurred at a pH of 5 and 32 degrees C. At 24 hr, the peak free lysine level, 120 mg/liter, is three times as great as the uncontrolled pH counterpart. In terms of total L-lysine (free plus protein-bound) the peak represents a 25% improvement over the uncontrolled case, based on an average 3.5% lysine level per cell weight. The greatest measured cell level, .9% by weight in the fermentation broth, or a 5 1/2-fold increase over th inoculum, was reached during the 36 degrees C and pH 3 run, while the largest measured ethanol value (3%, or 30% conversion by weight from glucose) was achieved during the 28 degrees C and pH 6 experiment. The optimal lysine run product, however, no less than 15% of the maximum cell and 30% of the maximum ethanol levels.     

Biogeographic comparisons of marine algal polyphenolics: evidence against a latitudinal trend

Summary Marine allelochemicals generally are present in greater quantity and diversity in tropical than in temperate regions. Marine algal polyphenolics have been reported as an apparent exception to this biogeographic trend, with literature values for phenolic concentrations significantly higher in temperate than in tropical brown algae. In contrast, our results, the first reported for Caribbean brown algae (orders Dictyotales and Fucales), show that many species have high phenolic levels. In addition, both our study and previous studies with north temperate and tropical species demonstrate that there is marked variation in algal phenolic levels within species from different locations. We conclude that high phenolic concentrations occur in species from both temperate and tropical regions, indicating that latitude alone is not a reasonable predictor of plant phenolic concentrations.     

An obligatory role of protein glycosylation in the life cycle of yeast cells

Two water-soluble carbodiimides, differing in molecular dimensions, have been used to characterize the cytochrome c binding site of bovine heart cytochrome c oxidase. Several polypeptide components of the enzyme contain acidic residues which are modified by these reagents. Carboxyl groups present in subunit II, VII and polypeptide c, are protected from modification when cytochrome c, equimolar to oxidase, is added and they can cross-link to the substrate once activated by the carbodiimide. Comparison of the modification patterns suggest that the most reactive residues are located on subunit II and VII, the former being also more exposed. The data obtained indicate that even though subunit II plays the major role in binding cytochrome c, at least two other lower Mr polypeptides contribute to the cytochrome c binding domain.     

Dolichyl phosphate linked sugars as intermediates in the synthesis of yeast mannoproteins: an in vivo study

Freshly prepared protoplasts of Saccharomyces cerevisiae X 2180 incorporate [³H]mannose and [¹⁴C]glucose for about 30 min into glycolipids and mannoproteins. Among the radioactive glycolipids formed dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides have been identified. The oligosaccharides released by weak acid from the dolichyl pyrophosphate were treated with endo-N-acetylglucosaminidase H and separated by gel filtration on Bio-Gel P-4. The largest oligosaccharide obtained corresponded exactly in size to Glc₃Man₉GlcNAc₁ the compound formed also in animal tissues. Other oligosaccharides released from dolichyl pyrophosphate in addition to the glucose containing ones were mainly Man₉GlcNAc₁ and Man₈GlcNAc₁. No mannosyl oligosaccharide corresponding in size to the total inner core region found in native mannoproteins could be detected in a lipid-bound form.The radioactive dolichyl pyrophosphate oligosaccharides were formed transiently; after 40 min only about 40% of the maximal radioactivity was observed in this fraction. In the presence of cycloheximide this decrease did not take place.It is concluded that the dolichol pathway of N-glycosylation of glycoproteins in yeast cells is very similar, if not identical, to the reaction sequence worked out for animal cells.Dedicated to Professor Dr. Otto Kandler on his 60th birthday     

Development of Dipetalonema viteae third-stage larvae (Nematoda: Filarioidea) in micropore chambers implanted into jirds, hamsters, normal and immunized mice

Development of third-stage larvae of Dipetalonema viteae within subcutaneously implanted micropore chambers proceeded in all hosts tested up to the fourth-stage larvae and occasionally to adolescent worms. In the jird the timing of development was comparable to a natural infection. Although the mouse is an insusceptible host, larval development could take place, but was very slow. Two intraperitoneal inoculations of living third-stage larvae into mice induced the production of antibodies against the larval cuticle and against common antigens. In such immune mice the development of third- and fourth-stage larvae within micropore chambers was significantly inhibited, larval mortality was increased, and the larval motility was impaired.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig.

Studies were carried out to investigate the effects of prostaglandins (PG) in vitro on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1 alpha or PGF2 alpha to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (delta A385-420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11 beta-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.