+, and Ca+
+
closely paralleled the effect of these ions on K
+
and N a +
transport in the intact cell. The ATPase activity is dependent on M g
+ +
and is markedly increased by Na+ plus K +
, though either ion alone has little effect; Ca+ + is inhibitory. Ouabain (gr-strophanthin) and other toxic cardiac glycosides, which abolish ion transport in intact erythro
cytes, inhibit the N a +
plus K+-stimulated ATPase activity. This complex picture does suggest that the ATPase activity is not due to a single enzyme but probably to a system of enzymes. Skou suggested that K
+
and Na+
differ in their loci of action (85), and the Hokins recent study (81) of the effect of Mg+ +, Na+ +, and K + on the diglyceride kinase and the phospha
tidic acid phosphatase suggests that the phosphatidic acid cycle could be responsible for the ATPase activity. If either or both of these enzymes were inhibited by Ca+ +, this would lend support to this interpretation, but no such studies have so far been reported. The case for the ATPase activity being due to the phosphatidic acid cycle has, however, been weakened by the brief report that ouabain stimulated the turnover of phosphatidic acid in brain slices (89). Ouabain had no effect, however, on phospholipid turnover in brain homogenates, and its effect on the diglyceride kinase and phosphatidic acid phosphatase of the erythrocyte membrane remains to be tested.
IV. ALTERNATIVE PATHWAY FOR LECITHIN SYNTHESIS;
TRANSMETHYLATION INVOLVING GLYCEROPHOSPHATIDES Following the observation of Hall and Nyc (90) that in a choline-de-ficient mutant of Neurospora crassa the lecithin normally present in this organism is replaced by a mixture of the phosphatidyl derivatives of mono-methylethanolamine and dimono-methylethanolamine, evidence has accumu
lated that lecithin may be synthesized by the methylation of phosphatidyl
ethanolamine and that this indeed may be a major pathway for the syn
thesis of lecithin in a wide range of animal tissues. Bremer, Figard, and Greenberg have presented evidence for the series of reactions (shown in Fig. 8) in rat-liver microsomes (91) by following the incorporation of C
u from /S-adenosylmethionine-CH3-C
14 .
5. PHOSPHOLIPIDS 207 Phosphatidy lse rine
Phosphatidylethanolamine Adenosylmethionine s
Ρ hosphatidy lmonome thy le t hanolamine Adenosylmethionine >
Phosphatidy ldime thy le thanolamine Adenosylmethionine ^
Lecithin
F I G . 8. Reactions involved in the synthesis of lecithin.
Further support for this reaction sequence has been provided by Gibson et al. {92), and Artom and Lofland have also demonstrated the last step in rat-liver microsomes (93). Exogenous phosphatidylethanolamine did not stimulate the incorporation of C
1 4
; either it is not able to reach the enzymic sites in the microsomes or the methylation reactions are confined to a specific endogenous lipid, possibly in a lipoprotein complex. The in
corporation of labeled C H3 groups into the microsomal lipid had a sur
prisingly high pH optimum at about 10, and the authors suggested that this may be due to the pi? of ethanolamine at 9.5, an uncharged amino group being required for reaction with the positively charged S-adenosyl-methionine.
The mechanism of the synthesis of phosphatidylserine is as yet un
known; however, Hubscher, et al. (94) have demonstrated the incorpora
tion of uniformly labeled serine into phosphatidylserine with rat-liver mitochondria. The incorporation had a pH optimum of 7.4 and was stimu
lated by Mg+
+
and more strongly by Ca+
+
(4 X 10~
3
M), although higher concentrations of the latter ion were inhibitory. CMP alone among the mononucleotides tested stimulated the incorporation, and C T P was with
out effect. As Ca + +
by virtue of its inhibition of both phosphatidic acid phosphatase and
diglyceride
transferase would probably block the synthesis of phosphatidylserine by a cytidine diphosphate serine pathway coupled with the fact that C T P did not stimulate the serine incorporation, it appears that phosphatidylserine is formed by another route. The role of CMP is obscure, and the lack of a requirement for an energy source sug
gests that it may be a simple exchange reaction.
208 J. Β. DAVENPORT
Dils and Hubscher have also demonstrated a Ca + +
-dependent incor
poration of (Me-C 1 4
) choline into lecithin in rat-liver microsomes (95).
They suggested that this was due to a reversal of phospholipase D activity.
They produced evidence that microsomes incubated in the presence of Ca+
+
contained rather more phosphatidic acid than a control, indicating phospholipase D activity. However, this might have been due to the effect of Ca
+ +
on diglyceride kinase and phosphatidic acid phosphatase activities known to be present in microsomes of other tissues (82). The phosphatidic acid phosphatase of chicken liver is inhibited by Ca+ + (64). The in
corporation of M e - C 14
in this case also had a high pH optimum (9.0), and it may be due to the methylation of phosphatidylethanolamine by the methylation pathway, the choline acting as a methionine precursor (96).
However, Bremer and Greenberg found that added choline did not affect the incorporation of labeled C H3 in their system. The point could probably be settled by studying the effect of substances known to inhibit the methyl
ation reactions on the choline incorporation system of Dils and Hubscher.
Organic mercury compounds and mercuric chloride strongly inhibited the methylation reactions, and the effect was reversed by glutathione (97), evidence strongly suggestive of the involvement of —SH groups. Mer-captoethanol and BAL, however, were inhibitory, but this has been shown to be due to their S-methylation by the 5-adenosylmethionine, thus di
verting the methyl groups from the lipid pathway (98). Highly specific inhibition is brought about by analogues of /S-adenosylmethionine, viz., /S-adenosylethionine (97) and iS-adenosylhomocysteine (92). These should be very suitable substances to test for their effect on the incorporation of choline into lecithin in the system of Dils and Hubscher and also should allow an evaluation of the contribution of the Cytidine diphosphate choline pathway to the synthesis of lecithin in systems in which both pathways may be operative.
The physiological significance of the methylation pathway for the synthesis of lecithin is not as yet apparent, but Gibson et al. (92) may well have emphasized an important aspect when they stated: "By the introduction of quarternary ammonium groups, the conversion of cephalin to lecithin would alter profoundly the charge on a lipid membrane and thereby change its structure or permeability. Such an alteration may play a part in determining the lamellar structure of the endoplasmic reticulum or in some of the functions of intracellular transport and con
duction which have been suggested for this component of the cytoplasm."
Against this viewpoint may be set the finding of Bremer and Greenberg (97) that the methylation pathway does not operate in brain tissue, a situation where variation in the charge on the endoplasmic reticulum may well be of great importance.
5. PHOSPHOLIPIDS 209 V. CONCLUSIONS
In the work surveyed in this chapter three types of inhibition of enzymic activity may be distinguished: (1) inhibition resulting from the establish
ment of colloidal repulsive forces which prevent the union of the enzyme and its substrate, (2) inhibition resulting from the disorganization of complex subcellular particles in which the enzyme is contained and the integrity of which structure may be necessary for enzymic activity, and (3) specific inhibition by reagents which probably react with the active center of the enzyme or with neighboring groups in the enzyme protein, e.g., —SH groups, essential for enzymic activity. Very little can be said at this stage about the third type of inhibition, as very few of the enzymes have been purified and nothing is known about their detailed structure nor about the detailed mechanism of the reactions they catalyze.
The first type of inhibition has, however, been placed on a firm experi
mental basis; its importance is obvious and its probable physiological significance is now apparent. The coalescence of plastids and lecithin emulsion particles observed microscopically by Kates and his qualitative explanation of the phenomenon (6, 14) were important observations of physicochemical conditions required for enzymic activity. However, the studies of Bangham and Dawson represent a valuable quantitative ap
proach to the problem of enzyme catalysis in heterogeneous systems, and have shown that important physicochemical parameters are the net electrostatic charges on the enzyme and on the substrate emulsion particle.
There is much evidence to suggest that this is not the only factor operat
ing; the attack by the phospholipase Β of P. notatum and by the phospho
lipase C of C. perfringens on low pressure films of lecithin without the addition of any activating molecule suggests that though charge relation
ships are important, other physicochemical factors also play a role. This is reflected under more physiological conditions by the ability of nega
tively charged phospholipase C to lyse well-washed human erythrocytes, which themselves carry a substantial negative charge (36). This may be due to a mosaic structure on the erythrocyte surface; and although the net charge of the erythrocyte is negative, there may be local areas carry
ing an excess of positive charge, loci at which the enzyme can attack the phospholipids of the cell membrane. Also significant in this regard is the fact that although the pure phospholipase C hardly attacks pure phospha
tidylethanolamine it readily does so when it is present in the erythrocyte membrane (62). It is obvious that a full understanding of such hetero
geneous enzymic catalyses depends upon a complete consideration of all the physicochemical factors operating at a surface or interface, of which the surface charge density and the orientation and spacing of molecules
210 J. Β. DAVENPORT
are probably the most important. The total amount of information and theory available in the fields of surface chemistry and colloid science may still not be adequate to explain many of the phenomena encountered in the living cell, but future advances in these fields and its application to the living cell must deepen our understanding of living processes. A great deal of work has been devoted to the study of the forces of interaction in lyo-phobic colloids, but much less is known about lyophilic colloids which involve, as well as charge relationships, binding forces such as hydrogen-bonding and a consideration of the organized structure of the water layers close to the colloid particles. An attempt in this chapter to explain a diverse range of observations on conditions controlling the hydrolytic activities of enzymes in heterogeneous systems in electrokinetic terms has met with only partial success, and there is no doubt that in many cases, although colloidal attractive and repulsive forces may be important, additional factors, such as specific inhibition or activation, involving the interaction of the active center of the enzyme with the hydrolyzable bond of the sub
strate molecule are of equal importance.
To separate the second type of inhibition, due to the disorganization of subcellular structures, from the first type may be an artificial subdivision.
It may be, for example, that the inability to demonstrate an enzymic activity in a soluble system derived from microsomes or mitochondria is due to conditions of assay; the optimum conditions for the demonstration of an enzyme contained in a lipoprotein organelle may well be very differ
ent from those necessary for its optimum activity as a soluble protein;
conditions which activate the particulate enzyme may inhibit it when it is soluble. The plant phospholipase D is a case in point, and it does em
phasize the importance of the environment of the enzyme in the living cell in controlling its activity. The failure so far to solubilize many of the phospholipid-synthesizing enzymes may be due to the use of conditions of assay which have not detected appreciable soluble activity or may reflect either a complete dependence of the activities of the enzymes on their structural and spatial interrelationships (maintained by the molecular structure of the organelle in which they are contained). It could also indi
cate that they are extremely unstable proteins whose tertiary structures lean heavily on their lipoprotein environment. Once removed from this pro
tective environment they may be easily denatured and inactivated. The importance of the molecular environment offered by subcellular particles and cell membranes to both enzymes and substrates highlights the neces
sity for a much more complete understanding of the nature and structure of lipoproteins, a field so far comparatively neglected and one which will receive increasing attention in the future.
The fact that many enzymes acting on water-soluble substrates are also present in lipoprotein membranous structures indicates that some of the
5. PHOSPHOLIPIDS 211 principles discussed in this chapter will be of significance in the regulation and control of the activities of these enzymes. Hammes and Alberty (99) have discussed theoretically the case of a small soluble substrate diffusing towards the active center (with its appropriate charge) of an enzyme. The approach of small water-soluble substrates to an enzyme contained in a membrane will undoubtedly be governed by the physicochemical condi
tions on the membrane surface, and this may be an important aspect of metabolic control in the living cell. In a consideration of ionic environment in the vicinity of a charged membrane it must not be forgotten that the principles discussed in this chapter apply also to hydrogen ions, and hence the pH on the surface of membranes will differ appreciably from that of the bulk phase. This phenomenon, first discovered by Peters in 1931 (100), was later emphasized by Danielli (101) in 1937 and has been discussed recently, with many pertinent examples, by McLaren (102). The studies by Bangham and Dawson of the electrokinetic factors governing the attack by a pure enzyme on a pure substrate emulsion (86), although far removed from and much simpler than physiological conditions, is nevertheless of great methodological and theoretical significancee, as it does allow the evaluation of physicochemical parameters, which may be of great importance in metabolic control. It is to be hoped that, in the future, kinetic studies of such heterogeneous systems will be undertaken and the kinetic data correlated with the interfacial physicochemical conditions.
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