Recent advances in our understanding of the role of inositides in intracellular signalling (Michell et al., 1981; Berridge, 1984a; Berridge and Irvine, 1984) have highlighted the need to understand the mechanisms by which the cellular levels of inositides, and hence indirectly of the second messengers generated from them, are controlled. A stimulation of PtdInsP 2 phosphodiesterase is the likely initial event occurring after cell activation by a large number of agonists (Fig. 1). As a result of phosphodiesteratic cleavage of PtdInsP 2 the two second messengers, diacylglycerol (Nishizuka, 1984) and InsP 3 (Berridge, 1984a; Berridge and Irvine, 1984), are formed. The former activates protein kinase C (Nishizuka, 1984) and the latter primarily mobilizes intracellular calcium (Berridge and Irvine, 1984), and these two pathways frequently, but not always (Labarca et al., 1984; Danthurluri and Deth, 1984), act synergistically to produce a final cell activation (Kaibuchi et al., 1984; Rink et al., 1983; Putney et al., 1984). Although the acute control of the levels of these intracellular messengers lies in the regulation of PtdInsP 2 phosphodiesterase activity, the regulation of diacylglycerol and InsP 3 over the longer term (after the first few seconds of stimulation) lies in the hands of the enzymes which generate PtdInsP 2, and those which degrade diacylglycerol and InsP 3. It is the possible mechanism for regulation of these enzymes which are discussed here.

The properties and control mechanisms of the enzymes involved in phosphoinositide catabolism are reviewed, in particular those of the PIP2 phosphodiesterase of rat brain. We have previously shown (Irvine et al., 1984a) that under approximately physiological ionic conditions, this enzyme will not hydrolyze its substrate in a lipid mixture similar to that of the inner half of a plasma membrane; however, if the substrate presentation is changed, the enzyme can become very active. We have tried to characterize more precisely this change in substrate presentation by mixing the PIP2 substrate with dipalmitoyl phosphatidylethanolamine, pig-liver phosphatidylethanolamine, lysophosphatidylcholine, dioleoylglycerol and distearoylglycerol. Although these experiments confirm the profound effect that substrate structure can have on PIP2 phosphodiesterase, an explanation in terms of a “bilayer” versus “non-bilayer” configuration is too simplistic to explain the enzyme’s preferences. Furthermore, it may be that the head-groups of phospholipids adjacent to the PIP2 substrate can also directly affect the activity. We suggest that in vivo PIP2 phosphodiesterase is not controlled by calcium, nor by enzyme protein configuration, but solely by the manner in which the substrate is presented to the enzyme.

The hydrogenation of α-linolenic acid to stearic acid which occurs with the mixed bacteria of the sheep rumen can be demonstrated in vitro by mixing exponential phase cultures of only two species of rumen bacteria and incubating for a further period with α-linolenic acid. One bacterium must be able to hydrogenate α-linolenic acid to predominantly trans-octadec-11-enoic acid which is then used 11 substrate by a second bacterium. Cultures grown from small mixed inocula failed, with the exception of one pair of bacteria, to hydrogenate α-linolenic acid to stearic acid. The products from these cultures showed that one of the pair of bacteria had outgrown the other. For stearate production it was necessary to use inocula with a minimum number of cells rather than cells in a particular phase of growth. Two of the bacteria used, P2/2 and T344, after several years in pure culture show an increased isomerization of the octadecenoic acid products.

The hydrogenation of a range of double-bond positional and configurational octadecenoic acid isomers (cis (delta 2 and delta 4 to delta 13) and trans (delta 2 and delta 5 to delta 13] to stearic acid by a rumen Fusocillus sp. were examined. The cis and trans delta 5 to delta 13 isomers were all hydrogenated to some extent by late-log-phase cultures added to suspensions of individual isomers and incubated for a further 3 h. Of the cis-isomers, delta 5 to delta 11 (79-73% conversion to stearic acid) were the preferred substrates. delta 12-cis- (30%) and delta 13-cis-isomers (5%) were poorly hydrogenated. Of the trans-isomers, delta 8, delta 9 and delta 10 were 45% converted to stearic acid, the other isomers were poorly hydrogenated. These results are in agreement with less extensive studies using sheep rumen micro-organisms. When cultures were grown from small inocula in media containing individual isomers more extensive hydrogenation was found than with late-log-phase cultures. At 24 h, cis delta 2, delta 4 and delta 5 gave the highest conversions to stearic acid (90%) followed by the cis delta 6 to delta 12 and trans delta 8 to delta 10 isomers (approximately 75%), although at 6 and 12 h delta 9-trans gave higher yields of stearic acid than delta 9-cis, probably because the growth of the cis cultures showed a longer log-phase.

The hydrogenation of all the methylene-interrupted cis,cis-octadecadienoic acids was examined using pure cultures of six rumen bacteria able to hydrogenate linoleic acid to stearic acid or its immediate precursor, trans-11-octadecenoic acid, after first conjugating the linoleic acid to cis,trans-9,11-octadecadienoic acid. Only the delta 14-cis,17-cis-isomer was not hydrogenated by at least one of the bacteria and no evidence was found that conjugation was necessary before hydrogenation except for the delta 2-cis,5-cis- and delta 9-cis,12-cis-isomers. Several isomers were hydrogenated to an extent close to that achieved with linoleic acid (delta 9-cis,12-cis). Those bacteria only able to hydrogenate linoleic to trans-11-octadecenoic acid gave only octadecenoic acid products and those bacteria able to hydrogenate linoleic acid to stearic gave variable yields of octadecenoic acids and stearic acid except with the isomers delta 12-cis,15-cis and delta 13-cis,16-cis when only octadecenoic acids were detected. At the substrate levels used (20 micrograms/ml), both inhibition and stimulation of growth were found but no common pattern emerged, nor was the growth consistently related to the extent of hydrogenation.

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The major phospholipids of the anaerobic rumen phycomycete Piromonas communis were phosphatidylethanolamine (38%), phosphatidylcholine (26%) and phosphatidylinositol (13%); no sphingolipids, glycolipids, plasmalogens or phosphonyl lipids were detected. Free fatty acids, triacylglycerols, 1:2 diacylglycerols and a variable amount of 1:3 diacylglycerol were identified, as were minor amounts of squalene and a triterpenol which is probably tetrahymanol. Approximately half the fatty acids were straight chain, even 12 to 24 carbon, saturated acids, the remainder being even 16 to 24 carbon, mono-unsaturated fatty acids. The double bonds in all except the 16 carbon acid were in the omega 9 position. The unsaturation is introduced by a delta 9 desaturase which uses stearic acid as substrate and which does not use oxygen as a terminal electron acceptor. 14C from acetate and glucose was incorporated into the fatty acids of all complex lipids, as were lauric, myristic, palmitic, stearic and oleic acids. [14C]Choline was incorporated into phosphatidylcholine and [14C]ethanolamine into phosphatidylethanolamine and phosphatidylcholine. Label from [14C]serine was recovered in phosphatidylserine and phosphatidylethanolamine, but was not detected in phosphatidylcholine.

Two species of rumen bacteria that have been previously shown to partially hydrogenate alpha-linolenic acid have been examined for their ability to hydrogenate gamma-linolenic acid. Free gamma-linolenic acid is hydrogenated in vitro to stearic acid by a rumen Fusocillus sp. (N.C.I.B. 11026), but only to cis,trans-octadec-6,11-enoic acid by a Butyrivibrio sp. The sequential hydrogenations are preceded by a delta 12-cis-delta 11-trans isomerization identical with that observed in the hydrogenation of alpha-linolenic acid and linoleic acid.

Five strictly anaerobic bacteria able to hydrogenate unsaturated fatty acids were isolated from sheep rumen. One was characterized as Ruminococcus albus, two as Eubacterium spp. and two as Fusocillus spp., one of which is named as a new species. The Fusocillus organisms were able to hydrogenate oleic acid and linoleic acid to stearic acid, and linolenic acid to cis-octadec-15-enoic acid. The R. albus and the two Eubacteria did not hydrogenate oleic acid but converted linoleic and linolenic acids to a mixture of octadecenoic acids; trans-octadec-II-enoic acid predominated but several isomeric cis and trans octadecenoic acids were produced together with isomers of non-conjugated octadecadienoic acids. The intermediate and final products of hydrogenation by each organism were compatible with the results from mixed rumen bacteria.