WI I Ill 1 1 WIN MI I 4 ‘I _‘|N_.\ M II _cncnoo QR {Q3i§"E AN“ $3€Q$§§=€QLWE§ EABQL SM ”(’55 HQUSEIFL‘E' MUSCA EQMEST CA lWfl .4» m: :\ 3710.323 foo é‘im Dogma of M 3: MCL‘EEGM‘I STATE 3330123... $3 Rohext Ham! :V’iiéiay. 352'. 1969 THESib LIBRARY S Michigan State University ABSTRACT STUDIES ON IODINE AND PHOSPHOLIPID METABOLISM IN THE HOUSEFLY MUSCA DOMESTICA BY Robert Harry Millay, Jr. The products of iodine metabolism and experiments with an in vigrg iodination system are described. Products isolated by extraction and acid hydrolysis, followed by paper and Dowex column chromatography in- clude.monoiodotyrosine, diiodotyrosine, iodide, and iodine. A very small amount of.a slightly larger,.neutra1 iodine-containing compound was isolated by Sephadex chromatography of the water extracts. Attempts to demonstrate enzymatic iodinating activity in XEEEE were unsuccessful, as were attempts to show peroxidase activity. The presence of acyl-CoA-lySOphOSpholipid acyltransferase in the housefly larvae microsomes is confirmed and some prOperties of this and microsomal phospholipases are described. The acyltransferase activity. is dependent on acyl CoA concentration. It is slightly stimulated by magnesium ion, and inhibited by lauryl sulfate. A phospholipase which utilizes l-sn-acyl-phospholipids as a substrate is not greatly affected by magnesium or calcium ion. It is stimulated by low concentrations of lauryl sulfate (10-3g), but inhibited by higher concentrations. High sub- strate levels of lysolecithin apparently inhibit the enzyme. Experiments using the pH stat to measure phospholipase activity are also described. In these studies calcium ion appeared to increase phospholipase activity. STUDIES ON IODINE AND PHOSPHOLIPID METABOLISM IN THE HOUSEFLY MUSCA DOMESTICA BY Robert Harry Millay, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1969 TABLE OF CONTENTS LIST OF FIGURES LIST OF ABBREVIATIONS PART I INTRODUCTION Review of literature Statement of problem MATERIALS AND METHODS Rearing of flies Preparation of enzyme fractions Preparation of fly material for identification of iodinated compounds Hydrolysis and chromatography Preparation of labelled iodotyrosines Enzymic iodination reactions Counting techniques RESULTS DISCUSSION PART II INTRODUCTION Review of the literature ii iv 10 18 21 21 Statement of problem MATERIALS AND METHODS Rearing.of.larvae Preparation of substrates Preparation of enzymes Phospholipase reaction mixtures: pH stat Labelled phospholipid incubations RESULTS pH stat experiments DISCUSSION LIST OF REFERENCES APPENDIX STRUCTURES OF IMPORTANT COMPOUNDS iii 25 26 26 26 28 29 30 32 4O 53 57 62 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES PART I Elution pattern of larval hydrolysates on Dowex 50. Elution pattern on Sephadex 6-10 of phosphate buf- fer extracts. Peroxidase activities of various subcellular frac- tions derived from housefly larvae. PART II Time course of acylation and deacylation of lysole- cithin by housefly larvae microsomes. pH Optimum of microsomal acyl transferases. Effect of Ca++ and Mg++ on acyl transferase and lysolecithinase activity. Effect of lauryl sulfate on lysOphOSpholipase. Effect of lysolecithin concentrations on acyl transferase reaction. Effect of lysolecithin concentration of the lyso- phospholipase reaction. Effect of lySOphosphatidylethanolamine concentra- tions on lysophospholipase. Effect of palmityl CoA concentration on acyl transferase reaction. iv 12 12 15 34 36 38 41 43 45 47 49 LIST OF ABBREVIATIONS ATP Adenine-5' -triph.osphosphate CoA Coenzyme.A DEAE Diethylaminoethyl NADPH Reduced nicotinamide adenine dinucleotide phosphate PE PhOSphatidylethanolamine DIT Diiodotyrosine MIT Monoiodotyrosine PART 1 INTRODUCTION Review of literature: When iodine in the form of radioactive 1311- (iodide ion) is fed to some insects, it is fixed to carbon (1,2). No requirement has been found in insects for iodine, as there has in mammals, and no function has been suggested for iodine fixation in insects, except detoxification. The thyroid gland, or an analogue thereof, occurs in all verte- brates. The function of the gland is to produce thyroxine, which is a hormone that regulates the basal metabolism of the organism. Thyroxine is the result of the conjugation of two molecules of diiodotyrosine (DIT) with the loss of a molecule of alanine. It has been conjectured (3) that the thyroid of vertebrates evolved as a specialized and encap- sulated version of the iodine-concentrating structures of invertebrates, endowed with the capacity to digest the iodinated protein and release the iodinated amino acids into the bloodstream. Recently the enzymes responsible for the production of these iodi- nated amino acids in vertebrates have been isolated and studied. These were divided into soluble and particulate systems (4). The iodinating activity required molecular oxygen and cupric.ion. Hydrogen peroxide could replace oxygen if it was introduced in a suitably regulated way. Certain other compounds, such as isomeric tyrosines, N-acetyl derivatives, 2 and p-hydroxyphenylacetic acid were also iodinated, whereas certain other similar compounds inhibited iodination. This iodination was proved to be enzymatic. Several workers found that the particulate enzyme could be solu- blized and isolated by a combination of tryptic.digestion and.column chromatography (5,6,7). The enzymes from several sources were found to be hemoproteins of molecular weight 64,000 to.lO0,000.. They could complex with cyanide and other heme complexers and.were thus inactivated. These enzymes could iodinate tyrosine, thyroglobulin, and other proteins. They required H202, and probably used a reduced pyridine nucleotide (NADPH) as a hydrogen donor. In all respects they behaved like typi- cal peroxidases. Apparently the thyroglobulin molecule directed synthesis of thy- roxine by conjugation of DIT residues (8,9), but the peroxidase would catalyze the formation of iodotyrosines in any protein, or free tyro- sine. Indeed, it has been shown that a number of peroxidases of non- thyroid origin could perform this reaction. Among these were chlorOper- oxidase, mye10peroxidase, and horse radish peroxidase (10,11). Iodine not excreted in the form of triiodothyronine or thyroxine was scavenged by a deiodinase system. As described in sheep, this was an enzyme requiring NADPH and inhibited by cupric ion and para-mercuri- chlorobenzoate (12). In this way the thyroid could make efficient use of its small quantity of iodine. Iodine metabolism in invertebrates is much less thoroughly under- stood. Iodine uptake and concentration was found in a species of 3 DrosoPhila (l). Iodine was concentrated in the cuticular parts of the larvae, the puparium, and the adult exoskeleton. Separated larval 1311' from solution, although the puparium skins would not concentrate would. Iodine uptake was studied in.a.variety of insects by Limpel (2), who reported the formation of iodotyrosines,.triiodothyronine, thyro- xine, iodohistidine, iodine, and other unrecognized products from.iodide. The different insects produced widely differing amounts of.the.various compounds, so that products typical of one species might be found scarcely or not at all in another. The iodine was concentrated to a greater degree in the exoskeletons, than any other.organ of the insects studied. In studies on the.cockroach Peripaneta americana (2), it was found that iodine circulated as iodide and monoiodo tyrosine (MIT), and was secreted mainly as monoiodohistidine (MIH). It remained in the muscle as MIT, DIT and thyroxine, and was found as MIT in the cuticle. Studies on other invertebrates showed that some have the ability to concentrate radioiodide. Among these, the barnacle concentrated radioiodide in the horny foot region, certain mollusca concentrated 1311 in cu- it in peripheral structures, and an annelid concentrated ticular structures, much like the insects (13). The fact that thyroxine may be found among the products of insect iodine metabolism is not so surprising if one considers that non- specific peroxidases can initiate thyroxine synthesis in various pro- teins. Although it has been reported that thyroid extract has the 4 ability to increase respiration in insect eggs, this effect is appar- ently not to be generalized to other insect organs (14,15). So we may conclude that iodide incorporating systems in insects are not for the purpose of hormone production. Statement pf problem: The problem presented herein for the housefly is what are the products of iodine metabolism, and how are these products produced. The following experiments describe the products of iodine metabolism in the housefly, and also indicate the presence of an unidentified iodination product, present in very small quantities in the housefly larvae. MATERIALS and METHODS Rearing.g£.flies: The housefly, Mnsca domestica L., a DDT resistant strain, was kept in screen cages.at 300 C. They were watered and fed on a diet containing sucrose, dry milk, and DDT (l,l—p-chlorophenyl~2,2,2-tri- chloroethane) in the proportions.100:100:l., Larvae for the study were reared aseptically in cotton and cheesecloth-stOppered flasks on medium similar to that described by Monroe (16). For i2_!i!g studies about 0.2 m0. 131 I, supplied by Tracerlab, Waltham, Massachusetts, was added to the food. Preparation gf enzyme fractions: For the enzymatic studies non-radioactive, aseptically reared larvae were isolated by rinsing them free of growth medium. They were then ground in a Waring blendor with 0.21! sucrose, 0.02511 Tris HCL (pH 7.2), for two successive 15 second periods. The liquified larvae were squeezed through eight layers of cheesecloth and centrifuged. Particulate fractions were isolated by centrifugation at the following levels: 1000 X g for 10 minutes; 8000 X g for 10 minutes; and 40000 X g for 20 minutes. These fractions were labelled the debris, the mitochondrial fraction, and the lysosomal (containing heavy microsomes) fraction, respectively. The supernatant from the centrifugation was 6 also used. All Operations were carried out at 40 C. Preparation 2f fly material for identification 2: iodinated compounds: Flies and larvae which were reared on diets containing 1311 were fractionated by grinding in a Waring blender. The first extraction was done with chloroform or with Efbutanol: 0.1MUHCl (prepared by shaking butanol with an equal.part of 0.1M_HC1 and separating the upper phase), or with 0.1M phosphate buffer (pH 7.8) containing 1mg KI and Na28203. The liquified material was filtered through cheesecloth, which removed the chitinous parts, and separated into solvent soluble fractions, water soluble, and insoluble portions. The portions thus separated were examined for 131I-containingicompounds by.chromatography or hydrolysis and chromatography. The fraction soluble in phOSphate buffer was used in the Sephadex chromatography. Hydrolysis and Chromatography: The insoluble proteins and chitinous parts were subjected to many treatments to extract iodine-containing components, but the presence of oxidizing agents or strong nucleOphiles tended to degrade these components into iodide and other products. The most satisfactory technique was closed tube hydrolysis in lN_HCl at 1100 C for 24 hours. This did not completely break down the chitinous parts, but released enough intact iodo-compounds to identify. The supernatants from these treatments were chromatographed by the ascending technique on Whatman No. 1 filter paper, and were also subjected to column chromatography by the method of Christianson, gt El' (18). This was done with l X 60 cm 7 jacketed column of Dowex 50. The temperature of this column was con- trolled by.directing water from a.constant temperature bath through . the jacket. Elutions were performed with citrate buffers, 0.2M_in Na+, pH 3.25, at 30° c, and.0.35M in Na+, 13:15.28, at 50° c. Chromatography of the products was undertaken in several solvent systems in order to identify the iodinated compounds. Collidine and water (125: 44) with an ammonia atmosphere.proved to be the most satisfactory. l-Butanol: 2M acetic acid, made by shaking butanol with 2M acetic acid and separating the butanol phase for use, and ethanol: 0.2M ammonium carbonate were also used. The phOSphate buffer extracts for Sephadex chromatographvaere treated with trichloroacetic acid (to 10%) and filtered. This solution was extracted twice with ether and lyOphilized. The dried material was taken up in a small amount of water, containing a trace of Na28203, and applied to a column of Sephadex G-10 (4 X 28 cm). Column effluent was analyzed for radioactivity. Preparation 2f labelled iodotyrosines: 131 I-labelled iodotyrosines were prepared in a method similar to that of Brown and Reith (17). In a thin polyethylene tube attached to a syringe aliquots of carrier-free 1311, 0.1% chloramine-T in 0.1M borate buffer, pH 8.0 (25p1), L-tyrosine (0.01%, 10p1), and 25pl dis- tilled water were taken up, with a short bubble spacer between each. This was rapidly injected into a small vessel in a 360 C bath and held there 5 minutes. The reaction was then stOpped with 0.1 ml of very dilute NaHSO3 and an aliquot chromatographed to determine its identity. Enzymic iodination reactions: Incubations were done typically with 0.1 ml KI (0.005! with 1311 tracer added), 0.1 ml of larval protein, 0.05 ml lngCl, and enough 0.05M Tris HCl buffer (pH 7.4) to bring-the volume up to 1.0 m1. KCl was added to prevent non—enzymatic iodination. Reactions were incubated at 300 C for 15 minutes, whereupon the proteins.were preci- pitated with trichloroacetic acid and removed by centrifugation. The bulk of the inorganic.iodide-could-not be precipitated with an excess of AgNO3 and removed“ When.tyrosine was incubated.with the reaction mixture, the acidic conditions.prevented.its precipitation by the ,silver ion. However, the presence.of.protein interfered with the silver halide.precipitation, so that acid precipitation of the protein always preceded the AgNO‘,3 treatmentr. The proteins were subjected to closed tube HCl hydrolysis or trypsin-pancreatin treatment. The re- sulting solutions, along with the supernatants from the reaction mixtures, were applied to small (1 X 3 cm) Dowex 1 columns (200-400 mesh, acetate form). MIT was eluted by 30 ml of 0.025% acetic acid, and DIT was removed by 30 ml of 1% acetic acid (19). Free iodide ion remained tightly bound to the Dowex. Incubations were done with tyro- sine added (0.1 ml, 0.01M), with freshly prepared ascorbate buffer (pH 7.5, 0.025M), and with glucose (to 0.01M) and glucose oxidase added. Peroxidase activity was measured as outlined in the Worthington enzymes catalogue. To one of two identical 3 ml portions of 0.01! phOSphate (pH 7.0) containing 0.003% hydrogen peroxide and about 0.008% o-dianisidine, the enzyme (0.1 ml) is added, and the color difference is I 9. measured spectrOphotometrically at 460 my. Counting techniques:. Material from flies was dried on planchettes and counted in a Nuclear Chicago thin-window, low-background, gaSPflow planchet counter. Clear effluents from columns could be counted without further prepara- tion by Cerenkov radiation in a Packard Tri-carb scintillation counter. Paper chromatograms were cut lengthwise into strips and scanned for radioactivity on a Packard strip scanner. RESULTS The distribution of 1311 in a typical fractionation showed.most of the counts in the insoluble fraction, and most of the rest in protein or water soluble fractions. Less than one percent was lipid soluble. Subsequent analyses by chromatography showed that the lipid soluble material was probably molecular iodine. The water soluble counts were largely uncombined iodide, although some MIT was present. Radioactivity extractable from KOH soluble and the insoluble fractions by HCl hy- drolysis were MIT and DIT. The distribution of radioactivity was not appreciably altered by rearing the flies on labelled iodotyrosines rather than on iodide ion, although DIT was found in the water-soluble fraction. No evidence for other labelled compounds was found. Larvae aseptically reared in 131 I—containing,medium, showed a similar propensity toward concentration of radioactivity in their cuticular skins. After pupation, these larvae could be removed from the medium and the adults allowed to emerge. The empty puparia thus obtained, contained very high amounts of radioactivity and the flies which emerged, contained very little. A large part of this activity was fixed in the cuticle. Hydrochloric acid hydrolysis revealed the same iodotyrosines in the pupal cases and larval skins. Figure l is a graph of the eluent from a long (1 X 45 cm) Dowex 50 11 column thrdugh which the HCl hydrolysis material was.run. Half a liter of citrate buffer was used at 300 C, then the jacket temperature was brought to 500 C.and a liter of the second buffer was used. The first three peaks corresponded to iodide, MIT, and DIT, respectively. The other two smaller peaks were not identified, but occurred remote from most amino acids. Figure 2 shows the results of the Sephadex chromatography of the water extractable, non-proteinaceous larval material. The two large peaks were iodide as determined by paper chromatography, and confirmed by spraying with.a ferric nitrate hydrogen peroxide indicator. The first peak always came off the column at the end of the void volume; ELEL’ it appears that its molecular weight is larger than the exclusion value for G-lO - about 750 to 1000. Chromatography in either collidine or butanol-acetic acid-water solvents failed to move the substance from the origin. It was apparently unaffected by treatment with pancreatin. The substance was subjected to high voltage electrOphoresis (1400 v. at pH 3.9) with no detectable migration. When the substance was dialysed for 24 hours against distilled water, it was found largely in the dialysis medium. Finally, it was not retarded by either Dowex l or Dowex 50. The procedure worked out for the in_xi££g iodination assays pro- vided for ease of detection of iodinated products when inorganic iodide was removed by the procedures stated in Methods. The precipitated protein and the deiodinated solution could be much more easily examined. The protein thus precipitated would be contaminated with loosely bound 12 Figure 1: Elution pattern of larval hydrolysates on Dowex 50. The unfilled baseline represents the elution at 300 C with citrate buffer, 0.2M in Na+, with pH 3.25. The filled baseline represents elution at 500 C with citrate buffer 0.35M in Na+, pH 5.28. Peaks 1,2, and 3 correspond to I-, MIT, and DIT, reSpectively. Figure 2: Elution pattern on Sephadex 6-10 of phOSphate buffer ex- tracts. Elution was with distilled water. The first small peak is the unknown compound, described in Results. The next two peaks were I'. CPIVI L I50- I 353W .‘ l, ‘. ‘+ g L I00 200 300 FRACTION NO. CPM 300l 200- noor IO 2‘0 30 FRACTION NO. Figure 2 14 iodide, however. At first the technique of phenol solution of the pro- tein and washing with salt/carrier iodide solutions was tried. Then the technique of the Dowex purification was used. After HCl hydrolysis, this protein often showed the presence of MIT. Blanks, however, showed that non-enzymatic iodination was taking place on free tyrosine under the same conditions. At the suggestion of Robert Igo, various amounts of KCl were added to keep non-enzymatic iodinations at a minimum. The final effective concentration arrived at was 0.05M} Also, in experiments with peroxidase mediated iodination, it had been reported that similar concentrations of KCl were stimulatory to enzymatic iodinations (10). At this concentration of KCl non-enzymatic iodination was abolished, but no enzymatic iodination could be detected. It was reasonable to assume that the likely iodinating enzyme was a peroxidase of some sort. Therefore, attempts were made to stimulate its activity by running the reaction in ascorbate buffer, which under catalytic conditions, causes the formation of hydrogen per- oxide from oxygen. Also a hydrogen peroxide generating system of glucose plus glucose oxidase was introduced, both without effect. However, it could not be certain that the systems so added were functioning prOper- 1y. It was then decided to ascertain whether my fractions had measurable peroxidase activity. The Worthington assay adOpted was quite sensitive, as shown by the horseradish peroxidase control example in Figure 3. With this assay, however, no activity could be demonstrated in any of the fractions tested, except possibly a very small amount in the 1000 X g 15 Figure 3: Peroxidase activities of various subcellular fractions de- rived from housefly larvae. The assay is described in the Methods. Supernatant activity,H; 40000 g,I—., frozen supernatant, H; cell debris, [ll—4:]; supernatant plus horseradish peroxi- dase (portion after break shows results of addition of H202),O-—-O; horseradish peroxidase , H . A B'i-ORBANCE: 460 Mp 0.8 - 0.4 - ‘ l H l6 so so 90’ ISO TIME (SEC) Figure 3 l7 fraction (this is an extremely difficult fraction from which to iso- late an enzyme). All the starting O.D.'s could be attributed to tur- bulence introduced by the enzymes. In order to be sure that peroxidase activity was not being inhibi- ted by the presence of substances in the fractions, such as catalase, which competes for the hydrogen peroxide, a control containing both horseradish peroxidase and supernatant was run (supernatant has the highest concentration of catalase). The result was that activity was still detectable, although it.declined rapidly. Addition of hydrogen peroxide to this system restored activity, demonstrating that competi- tion for the H202 was the factor reaponsible for the loss of activity. This implicates catalase as the responsible substance. DISCUSSION The housefly seemed to be typical with respect to iodine metabolism in comparison to other insects. The main products of iodination were MIT and DIT. Besides iodide, the other products were obtained in ex- tremely small amounts, and were not identified. Since tyrosine is a precursor to the quinone-like compounds which are responsible.for the hardening.and darkening.reaction in pupation and in the adult fly, it was hoped to link iodine incorporation into the cuticle with this event. It has been reported that high concen- trations of iodide in the diet of Drosophila gibberosa had no apparent effect on the hardening and darkening reactions of pupation (1). Furthermore, the fact that the untanned larval cuticle concentrated iodine in organically bound form as effectively as the tanned adult cuticle seemed to indicate that another mechanism was implicated. The cuticular tissue contained a large amount of tyrosine, and it seemed likely that this was the site of concentration of the iodine. Other tissues also contained tyrosine including the blood, but they did not share the marked tendency to concentrate the iodine that the cuticle had. One explanation was that the enzyme responsible for iodination was loca- lized, adjacent to or in the cuticular tissues. It was conceivable that the shunting of such large amounts of iodine into a quiescent, 19 non-circulating form such as this had adaptive value. Iodine, like most rare elements of the earth's crust is unevenly distributed, and it is likely that in many places the insect gets along with practically no iodide in its food sources, while in other places it must cape with re- latively large amounts. If the iodide were oxidized to elemental iodine, a potentially dangerous chemical would be released into the tissues. Only oxygen.and hydrogen peroxide among naturally occurring compounds in living organisms have shown higher oxidizing.potential than iodine (21' 12': +.535 v.).(10). Organisms have elaborate systems for regu- lating.oxygen.in.tissues, and have more than one enzyme devoted to destruction of hydrogen.peroxide. It seems likely then, that organisms have deveIOped.such enzyme systems to.c0pe with the presence of iodine. One interesting.aspect was the nature of the compound(s) obtained by the gel filtration experiments (I use the possible plural since this is hinted at by the broadness of the peak eluted from the Sephadex at such an early stage). It was apparently large enough to pass through the 6-10 unretarded, but not so large that it was non-dialysable. It was not hydrolyzed by proteases, and was electrically neutral. One possibility was that it was some kind of large conjugated polyphenolic compound which had either been iodinated or had been constructed of iodinated phenols. However, experience has shown such compounds are ad- sorbed by the resin backbone of the Dowex, and thus retarded. Since all the iodine fixing enzymes described to date are peroxi- dases, one might suspect a similar situation in insects. Klebanoff has made the generalization that all such enzymes were peroxidases (11,20). 20 The possibility could not be ruled out that other types of enzymes were responsible for the iodine fixation reaction in the housefly. In fact, the evidence was that enzymatic iodination did go on in the fly, in spite of the absence of demonstrable peroxidase activity. Possibly the localized character of the enzyme made it difficult to find in the tissue fraction, since the cuticular parts were removed before the tissue was fractionated. Also, perhaps inhibitory factors were present in the homogenates.. Nevertheless, the negative.character.of these re- sults led me to switch to work on a second project, described in Part Two of this thesis. PART I I INTRODUCTION Review g£_the literature: The subject of phOSpholipases has been reviewed recently (21,22,23, 24,25 ). The phospholipases which are relevant to this thesis are the phosphatide acyl-hydrolases (EC 3.1.1.4) and the so-called lysolecithin acyl hydrolase (EC 3.1.1.5). A-type phOSpholipases, that is, the en- zymes which cleave the acyl groups from either the l or the 2 position of phospholipids are wideSpread in nature;' they occur in snake venoms and bee venoms (22), and in subcellular fractions of mammalian organs, such as liver, pancreas, Spleen, and brain (23,24,25) and in blood cells (23). The phospholipases from snake and bee venoms are soluble enzymes and are all of the phOSphlipase A type (22). It has been reported that lysOphospholipase activity exists in snake venoms (23), but is active only at pH's above 8. Snake venom phospholipase A activity is enhanced by calcium ions, and inhibited by fatty acids and some detergents. Lysolecithin reportedly increases activity. Sulfhydryl reagents in general do not affect the activity (22). Recently the enzymes in SEE; talus adamanteus venom (49) and Naja_naja_venom (48) have been purified. The two enzymes crystallized from Crotalus venom appear to be nearly identical in physical preperties except for electrophoretic and purification 22 prOperties. Naja venom contains probably more than five separable phospholipases. In rat liver van Deenen and his coworkers (26,27) have found phos- pholipase A activity associated with the mitochondria, A1 associated 2 with the microsomes, and lysOphospholipase in the supernatant fraction, as.well as a mitochondrial lipase. The phospholipase A2 also required divalent metal ion, the lipase was sensitive to paggfmercurichloroben- zoate, and the lysophospholipase was sensitive to detergent and heat. The phospholipase A seemed to be more active toward phosphatidyletha-d nolamine than lecithin (28,29). DeDuve (50) has found indications of phospholipase A and lysOphospholipase activity in rat liver lysosomes. . Phospholipase A occurs in porcine pancreas as a proenzyme which is activated by trypsin (51). This enzyme has been partially purified, and the active species appears to be identical with a previously iso- lated form. LysoPhospholipase has been reported in Escherichia coli (32), in rat liver and in rat brain (52). The latter enzyme was partially puri- fied from cell debris. Its pH optimum is from 7.2 to 8.6, and it was inhibited by p-hydroxymercuribenzoate, by detergent and by fatty acids. Besides hydrolysis, lysolecithin could be eliminated by a transesteri- fication reaction wherein two lysolecithins reacted to make one leci- thin and one glycerOphosphorylcholine molecule. This reaction was first discovered by Lands (33) and was subsequently demonstrated in soluble fractions from baker's yeast (34) and in polymorphonuclear leukocytes (35). In the latter cells, lysolecithin was acylated by other means in the 23 particulate fraction at low concentrations, the soluble enzyme acylated at higher concentrations of lysolecithin. It was reported that lysolecithin could arise from lecithin by transesterification with cholesterol. In rat liver, the particulate fraction esterfied cholesterol using fatty acid, ATP, and CoA, whereas the soluble fraction transfered an acyl group from lecithin to chol- esterol (36). Both phOSpholipase A and lysophOSpholipase have been reported in the housefly (37). Both enzymes worked on both the ethanolamine- and the choline-containing phospholipids. The phospholipase A activity was reported to require calcium ion, while the lysOphospholipase did not. The same enzymes have been found in the larvae of the mosquito £2125, pipiens (53). These were most active at pH 9.2. The phospholipase A ++ and Zn++. required deoxycholate, and was inhibited by Ca++, Hg++, Co The fate of lysolecithin is not determined entirely by phospholi- pases, as indicated above. Acylating enzymes occur which require CoA and ATP as well as fatty acids. Such enzymes acylate lysolecithin, lysOphosphatidylethanolamine, and other lyso-derivatives of phospholi- pids. These were first extensively studied by Lands (21). The enzymes catalyzing the acyl transfer esterified certain types of fatty acids preferentially depending on the position of the acceptor site of the lysophospholipid, and, furthermore, the relative efficiency of esterifi- cation was affected by the type of base in the lysolipid (21,38,41 ). The mechanism of recognition was obscure. The enzyme(s) responsible for esterfying the 2-position did so preferentially with unsaturated 24 fatty acids of the naturally occuring types (21), while Eggng-unsaturated fatty acids were esterified.preferentially at the l-position, as were saturated fatty acids (21,39). Enzyme specificity at the 2-position may have been due, in part, to dispersive forces in the unsaturated region of the fatty acids (47). It is now well established that natural phospholipids are usually very assymmetric with respect to distribution of saturated and unsatura- ted fatty acids (23). The pathways of phospholipid dg_nggg synthesis are well known (23,40), starting with acylation of glycerol-3-phosphate to phosphatidic acid, which is converted to diglyceride, which is then acylated to triglyceride or converted to phOSpholipid. The key inter- mediate here is phosphatidic acid, which ideally should show this asym- metric distribution of fatty acids. Lands and Hart (38) showed that phosphatidic acid in guinea.pig liver was almost randomly acylated, al— though rat liver showed more specificity. However, it has been stated that 92 page synthesis of phosphatides could not account for the special distributions of fatty acids in each phosphatide. This made the acyl transferase a good choice for the agent which provided this asymmetry. This argument has been challenged recently by van Deenen (41) using the fact that rat-liver microsomes acylated phosphatidic acid more specifically than did guinea pig-liver microsomes. His data showed, how- ever, that rat-liver phosphatidyltholine and phOSphatidyl ethanolamine have quite different fatty acid contents from phosphatidic acid, as well as from each other. He does not attempt to explain the discrepancy in guinea pigs. 25 Phosphatide acyl transferase has been reported in brain (42), and bacteria (32). So far it has not been reported in insects. Statement g£_problem: In order for the theories of Lands to be acceptable, the general na- ture andeidespre d appearance of the transacylase enzymes should be demonstrated. The purpose of this part of the thesis is to show that the acyltransferase enzymes do exist in the larva of the housefly, and to show some properties of this enzyme. This thesis will also show some properties of the phospholipases in the housefly larva. To help put the importance of the transferase enzyme into perspective, the studies will be shown in comparison, so that an idea can be gotten as to how some of these systems interact with each other. MATERIALS and METHODS Rearing 2f larvae: Larvae of the housefly Muggg_domestica L. were reared aseptically at 30° C in cotton-cheesecloth stOppered flasks, using medium similar to that described by Monroe (16). When isotopically labelled lipids were desired, the inorganic phosphate content of the medium was lower- ed (a modified Wesson salts mixture being used).and supplemented by 32P phosphoric acid (generally about 0.5 mC per flask) obtained from New England Nuclear,.Boston, Massachusetts. Larvae thus reared were harvested after four days by rinsing them.free of media with distilled water and used immediately. Preparation g£_substrates: Unlabelled lipids were purified from commercial sources (azolectin from.Associated Concentrates, Woodside, New York, and egg lecithin from Nutritional Biochemicals, Cleveland, Ohio) or from larvae. Labelled lipids were isolated from 32 P-fed larvae. Lipids extracted from larvae were obtained by homogenizing the larvae with 10 times (larval weight/ solvent volume) of chloroform: methanol (C:M) 1:1 in a Waring blendor for two minutes, followed by suction filtration. This extraction was repeated twice again with C:M 2:1. The resulting yellowish extract was rinsed free of water-soluble phosphorus and other impurities by twice 27 washing with 0.2 volumes of 0.01M'Na3PO4, and the upper, water-contain- ing layer was siphoned off and discarded after each wash. The chloroform layer was evaporated under reduced pressure and low heat (30—400 C), taken up in 20 ml chloroform and filtered onto a sili- cic acid column for separation. Silicic acid (Bio 811 100-200 mesh, obtained from Bio Rad, Richmond, California) was made into a slurry with chloroform and about 100 ml of this was poured into a step-down column. This column consisted of four tiers, each about.150 mm.long,. and arranged so that the top tier was 26 mm in diameter, the next 20 mm, the third 14 mm, and the lowest 10 mm in diameter. This column gave good separation of the phospholipids, and was able to accomodate the same quantities of crude lipid as a column the same length and uniform- ly the same as the tap width. After application of the crude phospho- lipid mixture, it was.washed with about 50 ml of chloroform, followed by 100 m1 of acetone. The acetone removed neutral lipids loosely bound to the silicic acid, and probably also removed water. Elution continued with C:M.4:l (250 ml) in which acidic phospholipids such as cardiolipin, phosphatidyl glycerol, and phosphatidic acid, and the bulk of the phosphatidyl ethanolamine are removed. Then followed 100 ml of C:M 3:2, which removed phosphatidylserine and phOSphatidylinositol. Next 150 ml of C:M 1:4 eluted the phosphatidylcholine. A final rinse with 100 ml Methanol removed residual phospholipids. In order to further purify the lecithin and PE fractions, further column chromatography was utilized. PE was purified by washing it through DEAE acetate (Bio Rad) in C:M 1:1, followed by rechromatography over 28 silicic acid. Lecithin could be further purified by chromatography over silicic acid. When the radioactive effluent was monitored with a GeigereMUller tube, practically pure PE and lecithin samples could be obtained from the.first column separation by judicious cutting of the fractions. The lyso_derivatives.were.obtaineduhymtreatment-of the-purified lecithin or PE with Crotalus adamanteus venom (Sigma Biochemicals, St. Louis, Missouri). The reaction products were extracted by a modified Bligh-Dyer procedure (44), using five volumes of.C:M 2:1, and rinsing with 0.02MLMgCl2 (2/3 volume) and distilled water.(equal volume). The chloroform layer was then dried, taken up in a small amount of chloro- form, and applied to a silica gelPG plate (silica gel from Brinkman 4 Instruments, Westbury, New York about 3/4 mm thickness). After deve10p- ment in chloroform: methanol: water (C:M:W) 65: 35: 4 the bands were visualized with iodine vapor, scraped off and eluted in small columns with 100 m1 methanol. The labelled lyso-derivatives so isolated, showed a single peak upon TLC on a radiochromatogram scanner, correSponding to the pure compound. Phosphate assays were done by the method of Bart- lett (46). Purification of the commercial lipids was essentially the same as the column chromatographic procedures listed above. Preparation pf enzymes: Aseptically reared third instar larvae were isolated and ground in a Waring blendor with 0.1M sucrose, 0.05M_Tris, 0.001M EDTA buffer, pH 7.2 or 7.6. For each 15 g of larvae, 30 ml of buffer was used. After 29 two 15 second grindings, the liquid was squeezed through eight layers of cheesecloth and.then centrifuged in a Sorvall superspeed centrifuge. Centrifugations were done at.800 X g,.8000.X”g,-and-400001X-g to re- move debris, mitochondria, andulysosomes,.reapectively. The superna- tant liquid.was then centrifugedIat.88000-X.g for.one hour. .The resulting:microsomal.pellet-was-resuspended-either-in.Tris.buffer (0.05M, pH 7-2) or in distilled water and recentrifuged at.88000 X g for one hour.. This pellet was taken up in diatilled.water or in incu- bation buffer to be used as enzyme. -The microsomal suspension.used.was not tested for lysosomal impurities, .All procedures wereidone.at..4o C . or over ice. Protein concentration was determined on these suspensions by the method.oerowry.(45).., Phospholipase reaction mixtures: p§_stat: Reactions were run in a glass cuvette held.in a constant tempera- ture bath over a magnetic stirring device. Six ml of 0.1M’NaCl was put in the cuvette and allowed to come to temperature (300 C). The glass electrode and titration input tube were immersed in the solution and the recording pH stat was allowed to bring the solution to a set pH. Each addition which was then made was allowed to come.to pH equilabration before the recorded rate was noted. Typical reaction mixtures contained 0.5 ml of microsomes taken up in distilled water to average 8-12 mg protein per ml of solution, and 0.5 ml of a sonicated phospholipid pre- paration, generally containing 25 mg lipid per ml of solution. Other additives were 0.1 m1 of lM_CaCl and 0.1 ml of 0.1M_1auryl sulfate. 2 Exact compositions are noted with Results. 30 The titrations were done with 0.004M NaOH. “The pH stat was read .so.that the.exact amount of.base.added per increment.shown.on.the re. reorder could be.computed.- The amplifier's sensitivityeallowed detec- tion of about 0.05.units of pH change. The pH's used ranged from 6.5 to 8.0. Labelled phospholipid incubations: Typical incubations contained 0.2 ml imidazole buffer (0.2!, pH 6.5) which was 0.001M in.EDTA, 0.02 mole of palmityl CoA (Sigma Biochemicals, St. Louis, Missouri) usually added.in 0.2-ml water,.0-025 ml.0.2M MgClz, 0.025 ml lysolecithin .or.1yso PE. (from a 1pmole/ml sonicated suspension) and 0.025 ml microsomes (suspended in.buffer to about 4-6 mg/ml). Reactions were carried on for 20 minutes in conical centrifuge tubes in a shaking water bath at 300 C. Variations from this will be noted in the Results. At the end of incubation time the reactions were killed with 1.3 ml methanol, followed by 2.7 ml chloroform. This mixture was washed by vortexing with 0.5 ml 0.02M;Mg012, and the upper layer was transferred to a polyethylene counting vial. The chloroform layer was rinsed once again with 0.5 ml 0.02M,MgC12.and the water layer transferred to the same counting vials. The chloroform layers were evaporated under a stream of air, and the lipids dissolved in a few drops of C:M, 1:1 and applied to silica gel F plates (Merck, provided by Brinkman Instruments, West- bury, New York). After deve10pment in C:M!W 65: 35:4' the spots were visualized with iodine vapor or detected with a G-M counter. These spots were scraped off into separate counting vials. PrOperly done, this 31 method could account for.96% of the counts originally.added. All counting-was done.in a Packard.Tri-carb liquid scintillation counter, utilizing the Cerenkov.radiation produced by-the 32?. At first water was used as.a.scintillator, until it was.found.that meth- anol gave higher efficiency in.countingi--Afterwards, all the.silica gel scrapings were counted without added scintillator, utilizing the properties of the vial itself. RESULTS The original intent of this investigation was to find and charac- terize the enzymes catalyzing the transfer of acyl groups to lysophos- phatides from acyl CoAs. Both the 40000988000 x g microsomal pellet and supernatant were examined, and it was determined.that the.acylating activity was in the microsomes. No acylating activity was detected in the supernatant. Subsequent experiments.utilized.only the microsomal fraction for the enzyme reactions. It was quickly found that another enzyme was present in microsomes, which competed for the substrate.(g;g;rlysolecithin), breaking it down to fatty acid and glycerylphosphoryl base. Thus, one reaction had to be measured while another was inhibiting it,.by using up substrate needed for the first reaction. This situation was further complicated by the existance of a phospholipase.A in the microsomes (37), which breaks down the phospholipid made by the acyl-transferase, so that the problem of detection concerns both loss of substrate and loss of product. Under these conditions, unless the enzyme could be separated, or inhibited, it was thought that a study of their interaction would be the most fruit- ful approach. When different concentrations of enzyme were used, it was found that the activity of the enzymes did not increase with added enzyme. Indeed, 33 the transferase activity showed a decline.at.highermenzyme.concentra- tions. It was decided to.use a concentration.of enzyme.which-gave.optir mum transferase.activity. This was at.about 0s25 mg protein.per ml, Time course reactions (Figure 1) showed that-the rate of.reaction remained nearly linear for about.20 minutes. “Thereafter, 20 minute incubations were used.. The pH Optima Of the transferase.for both lysolecithin and lyso- phosphatidylethanolamine are shown in Figure.2. -These appear to be in the range of pH 6A7. The.lysolecithinase was more.active above pH 7, and this.may cause the transferase peaks to be biased toward the acidic pH. For the incubations, a pH was chosen which would.show the transfer- ase reaction to greatest advantage, pH 6.5., The effects of calcium and magnesium ions.are shown in Figure 3. There appeared to be a very slight stimulation by magnesium ion Of both the acyl transferase reaction and the lysOphosphOlipase reaction with the substrate lysolecithin. Calcium ions, on the other hand, may have stimulated the lysOphosphOlipase reaction somewhat, but appeared to in- hibit the acyl-transferase reaction. The stimulation, if any, of the former reaction may have been in part due to the inhibition of the latter. When the microsomal preparations were subjected to sonication, very little activity was removed, and the enzyme activity was lowered. The lysOphosphOlipase activity which was removed was inhibited markedly by calcium; ions. When the microsomes were frozen and stored several days, all transferase activity was lost, and the lysOphOSpholipase activi- ty was inhibited by both calcium and magnesium ion. 34 Figure 1; .Time course of acylation and deacylation of lysolecithin by housefly larvae.microsomes: Incubation mixtures contained 0.15 moles each of lysolecithin and palmityl.CoA, 0.8mg of microsomal pro- . tein, and enough 0.1MpTris,an.7.4.to.bring.the mixture up to 1.5 ml. Lecithin formed,O—O; water soluble counts formed,H . MpMOLES 35 TIME (MIN) Figure l 36 Figure 2: pH optimum of microsomal acyl transferases: Incubations were done as described in Methods except for buffer types, which were: 0.2! citrate, pH 5.4; 0.2M imidazole, pH 6.2 and 6.8; 0.1M imidazole, pH 7.4; 0.1M_Tris, pH 7.4; and 0.2M_Tris, pH 8.0 and 9.1. 37 gal CPM IN 40" LECITHIN 20L 5 A 7 9 pH t O 60 0 PM CIN 40 PE 0 20- 0 6 :z ' ‘ ‘ 93 pH Figure 2 38 Figure 3:. Effect of Ca++ and Mg++ on acyl transferase and lysoleci- thinase activity: Incubations were as described in Methods, buffer was 1 X 10’4M in EDTA, and appropriate additions of metal ion were substi- tuted for the MgClz. Substrate shown is lysophosphatidylcholine. GlycerOphosphorylchOline formed, H; Lecithin formed, O—O . MpMOLES MpIVIOLES 39 A A a 3) A A 2- I - 42. 0 so A A I I M . A Q n_ ,w in .v, v— IO so so CAH (MM) Figure 3 40 Figure 4 shows the effect of various concentrations of sodium lauryl sulfate on the lysolecithinase.reaction.-.All concentrations of lauryl sulfate inhibited.the.transacylase.reaction. The lysOphosphOli- pase reaction was stimulated by the detergent at low concentrations, and inhibited.at higher concentrations. The effects of.varyingmconcentrations.of.lysophOSphOlipid are shown in Figures.5, 6, and 7, for lysolecithin.and lysophosphatidylethanolamine. The acylation of lysophosphatidylethanolamine did occurr.but at.a rate which was poorly.competitive.to the breakdown.“ This transacylase also acylated lysophosphatidylqBLmethylcholine.at a rate more closely matching that of lysolecithin. From the data shown, it was attempted to obtain an apparent Km by means of reciprocal plots. This showed the lysophos- pholipase to have similar Km's for both lysolecithin (5.7 X 10'4M) and lysophosphatidylethanolamine (5.6_X 10'4M). Also the lysOphosphOlipase was apparently inhibited by higher concentrations of lysolecithin. The apparent Km for the acyl transferase with lysolecithin was 2.6 X lO'SM, much lower than for the phOSpholipase. The transferase reaction was dependant on acyl COA (Figure 8). The amount of palmityl COA needed for near maximal activity was less than half the amount of lysolecithin present. pg 5%; gper iments: The pH stat was tested for the eventual use in measuring the lyso- phospholipase and phospholipase reactions without the need of using la- belled substrates. These reactions released free fatty acids which could be titrated with NaOH, the amount used being a measure Of fatty acid 41 Figure-4:- Effect.of lauryl sulfate on lysophospholipase: Incubations were as described in Methods, with the addition of 0.025 ml aliquots Of freshly prepared sodium lauryl sulfate. Substrate is lysolecithin. MpMOLES _ 42 2o LAURYL SULFATE (M M) Figure 4 4O 43 Figure 5: Effect Of lysolecithin concentrations on acyl transferase reaction: Incubations were as described in Methods, except that various aliquot sizes of lysolecithin suspension were added, from 2 to 50 pl. each, with compensations made.in the amount of buffer to simplify arith- metic. Concentrations are similar units for the double reciprocal plot. The approximate Km calculated from this graph was 2.6 X 1073M. o.I2 one 0 [S] (M M) 2 44 0.64 8. . 4. O 0 Std ms. IE> Aziheozai >2 uo Figure 5 45 Figure 6: Effect of lysolecithin concentration of the.lysOphospholi- pase reaction. Incubations were as described in Methods, except various amounts of lysolecithin suspension, from 2Jpl to 200 pl were added, with concomitant changes in.buffer. Concentrations were in similar units for the double reciprocal plot. For this graph, the estimation of the Km was made on the basis of the three lowest concentrations, one of which fell outside the plot. This was done for the sake of ease of visualization Of the intercepts. The approximate Km obtained from this plot was 5.7 x 10'45. 46 (T A0. 0 O fi‘g v: 2’- I0 .‘\! O 00 8d) e m 'iOJd W All "13A “wyselowdw DO Figure 6 (MM) [5] 47 Figure 7: Effect Of lysophosphatidylethanolamine concentrations on lysophospholipase: Incubations were as described in the text, ex- cept that amounts of lyso PE suspension from 2 to 50 pl were added, with balancing changes.in the buffer. The double reciprocal plot was again magnified to make the intercepts apparent. The approximate Km derived from this plot was 5.6 X lO'AM. 48 ))30 0.6- / LA 0:4 ‘—‘—‘ .8 “'0 1 i IOJ W NM/Selomw AlIDO'IEIA Figure 7 0.2 (mil/J) [S] 49 Figure 8: Effect of palmityl COA concentration on acyl transferase re- action: Incubations were as described in Methods, except that various palmityl COA aliquots (in buffer) were added, balanced by amounts of buffer. Substrate used was lysolecithin. 50 40 9o a 9de ms. .25 >931: Econ? 6. 0 [S] (MM) Figure 8 51 cleavage. Theoretically, this was the reaction being tested on the pH stat. Since the amount of alkali used was readily-seen and plotted as the reaction proceeded,.the pH stat became valuable in following” the immediate.responses Of the system to additions.. Preliminary ex- periments were.performed with a mixture of phospholipidst(azolectin), .and seemed to.show.a stimulation-Of.releaserof-fatty acids by calcium. Various purified phospholipids.were.then used to try to ascertain their reactions with.calciummand-with each other.. . Blanks showed that the.microsomes incubated alone, had an en- dogenous rate of acid release. This.was stimulated by lauryl sulfate and by calcium, and also by phospholipid. -Some phospholipids.were better stimulators than others,.however. .On the average,.both lyso- lecithin and PE were better.stimu1ators of.acid release than leci- thin. Little or no acid release was shown without the presence Of microsomes. The effects of mixtures of these compounds on the acid-production rate could be generalized as follows: lauryl sulfate apparently inhi- bited acid release stimulated by lecithin, but had little result on that from PE. Olive oil stimulated acid release only in the presence of lauryl sulfate (this was Probably not due to added phospholipid, as the Olive oil used, contained about 0.3% phospholipid). Calcium stimulated acid release to.a greater degree in the presence of lecithin, but.not in the presence of PE. Lecithin and PE did not appreciably affect one another when incubated together, but their rates may not be altogether additive. On the other hand, there was a marked inhibition of acid release by 52 lysolecithin in the.presence.of.either lecithin or PE. These genrealizations have some amount of predictive value. One effect not predicted was that addition.of lecithin over a period of. time in small.aliquots did not lead to proportional increases in the rate of acid release. DISCUSSION According”tomthe-theorieswof-Lands-(21,38)xthe.enzymesiresponsi—. ble for the acylation of lysOphosphatides are the most important deters . minant as.to.the.molecular.apecies.of.the phosphatides present in the animal body- -If the distribution.of.therfatty acids inithe.phosph09. lipid.has.more than.general.structural significance (gpg;,.phosphatides with certain.acyl sidechains which contribute.to.enzyme action or speci- ficity), then it is.the acyl transferases which must be responsible for the efficiency of these reactions and for the structures. Such an enzyme occurs in houseflies, but much study must be done to deter- mine if its activity and specificity is of the same type as the rat enzymes. The theory of Lands indicates why lysolecithin, with its powerful detergent and lytic prOperties, should appear in the organism. It has been suggested (23) that it plays a role in the fragmentation and breakdown of membranes in the growing organism. Now it seems likely that lysolecithin must exist even in the absence of such disruptive processes, in order that prOper phosphatide fatty acid composition be maintained. The relative efficiencies of acylation and breakdown of the phos- phatides with respect to the base moiety was interesting. Phospha- tidylethanolamine is the predominant phospholipid in the housefly (43), 54 and conditions in.the microsomes apparently favor breakdown of the de- acylated lipid, with reacylation poorly competitive using.the incubation conditions described in the Methods. 'Phosphatidylcholine was much more efficiently.reacylated. The housefly can not.synthesize.choline, and so this efficiency.cou1d be a.lecithin conservation mechanism. The similar response to B-methylcholine .phosphatides -isconsistent with the prOperty of fl-methylcholine. to substitute for dietary choline in larvae. The differences in the apparent Km's of the acylating enzymes and the phospholipases was an indication that at low substrate levels, the acylation mechanism was working.at high efficiency, while the-hydro- lytic enzyme was relatively inactive. The inhibition.of the.phospholipase activity.hy high substrate.. concentrations has been seen before (30).. This could.be due to the detergent action of.the lysolecithin, however, a similar effect was not observed with lysOphosphatidylethanolamine.- The effect of higher substrate concentrations has also been noted for the acyl transferase (21), in which high concentrations of acyl COA are inhibitory. This might have been due primarily to the fact that at higher concentrations the acyl COA forms micelles, decreasing the apparent concentration Of the substrate. The problem of micelle formation with lipid substrates makes interpretation of some data difficult. For example, it renders the expression of concentration academic, since the individual molecules of lipid are part of a large group and not freely accessable. This makes Km's for enzymes working on these substrates unreliable and very hard to measure. 55 As shown.in Figure 8, the relationship.between.palmityl COA con- centration and transferase activity may be sigmoidalrmbut.this.could be a, due to.scatteringwof-the.pointsuat-lownreaction.rates, The pH stat results were difficult to.interpret. “Adequate controls were best made at the same time with the.same.enzyme preparation, espe- cially since the.crude microsomes.were subject.to.variation.in specific- activity and to loss of activity on standing..uFor.this reason,.a com-. pendium of control measurements was.made-n From this it was.obvious . that variation.existed from.experiment to.experiment...Despite.this, these generalized controls were capable.of predicting experimental re- sults. The measurements with the pH stat did reveal.some-problems.. One was why the pH stat showed an increase of acid production with lecithin as substrate, by addition of calcium, in Opposition with the results found by another worker in our laboratory.using.labelled lipid (S.S. Kumar, unpublished results). One possibility was that the increase was due entirely to stimulation of the endogenous rate of phospholipid hy- drolysis, not only by calcium, but by lecithin. Perhaps calcium was re- sponsible for a simultaneous increase in rate of turnover in microsomal lipids and decrease in the rate of exogenous breakdown. It could also be due to changes in the micellular type causing release of extra ions from a larger surface area of phospholipid. The effect of calcium on 32P the endogenous rate of breakdown could best be measured by using labelled microsomes, rather than exogenous substrate (28). The effect of lauryl sulfate on the endogenous rate was probably 56 due to its expanding.and.disrupting.effect on the microsomal membranes- Its effect on the exogenous lipids was probably their solubilization. The detergent character of lysolecithin may be.the factor.which allowed its greater apparent increase.in.acid production. .The inhibition due. to whole phospholipid.may have.been due to inhibition of the lysophospho- lipase or it may.be due to incorporation of the lysOesubstrate into the lipid micelles, removing it from reaction. Experiments still need to be done on inhibitors of the system. Perhaps this will bring to light a way to more effectively study the interrelationship between endogenous.and exogenous.enzymes of phospho- lipid metabolism in.microsomes. .Another approach is isolation of the enzymes Of phospholipid metabolism from-the.membranes, with their.sub- sequent purification.. The loss of unity.of the system.makes it diffi- cult to tell if the enzyme isolated, is behaving characteristically. The use of judiciously applied.inhibitors.and more carefully selected con- trols, then, appears to be the better method of solving the problems of the interactions among the phospholipids and the enzymes responsible for their metabolism, and the membranous structures which contain them both. LIST OF REFERENCES 10. 11. LIST OF REFERENCES Wheeler, B.M.: Halogen metabolism Of.Drosgphila_gibberosa I. Iodine metabolism studied by means ofIJ‘I. Jour. Exptl. Zool. 115, 83 (1950). Limpel, Lawrence E.: Iodine Metabolism in Insects. Thesis Univer- sity of Wisconsin, 1957. Gorbman, A.: Some aspects Of the comparative biochemistry of iodine utilization and the evolution of thyroidal function. Physiol. Rev., 32, 336 (1955). Serif, 6.8., and S. Kirkwood: Enzyme systems concerned with the synthesis of monoiodotyrosine. Jour. Biol. Chem. 233, 109 (1958). 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I , I 1- R‘O'CH R’O‘CH o I I + l H a. Intro-(5:-o-CH.,CII,-N(at)3 H.C-O«P-O~CII,CII,-I\IH1 LEC 1mm: PHOSPHATIDYIETHANOIAMINE PHOSPHATIDYLCHOLINE H, C- O- R H, C—oI—I , I I R-O-C'H o R-O-CH II + | H H,c-o—I‘>-o- CH-CH,-N(CH,) H1C-0-P-0'CH CH -N(CH ) I 3 I ‘ t 33 o_ ca, 0. PHOSPHATIDYLfCér-METHYLCHOLINE 2-ACYLGLYCEROLPHOSPHORYL_’- CHOLINE; Z-LYSOLECITHIN HmC‘O’R I HO-CH H,c-o- :O ‘O'CH.CH.:N(CH3)3 C3~1D 1sACYLGLYCEROLPHOSPHORYLCHOLINE; l-LYSOLECITHIN (R= fatty acyl group)