Preparation and characterization of total, rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats.

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Interaction of Ca 2+ with endoplasmic reticulum of rat liver: a standardized procedure for the isolation of rat liver microsomes

A standardized method has been developed for the rapid isolation of rat liver microsomes using Ca²⁺ and its advantages over other available methods have been outlined. In addition to hydrolytic enzymes and chemical composition, the important enzymes in the electron transport system were determined in Ca²⁺ microsomes and normal 105,000g microsomes and indicate only minor differences between the two preparations. Two classes of microsomes—smooth and rough particles prepared with or without the addition of Ca²⁺—were compared for their chemical and biochemical properties and indicated little differences within each microsomal fraction. The ability of other divalent cations like Mg²⁺, Fe²⁺, Ba²⁺, Zn²⁺, and Hg²⁺ to aggregate the microsomes was observed while the monovalent and trivalent cations tested did not appreciably sediment the microsomes under the present experimental conditions.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

A Biochemical and Morphological Study of Rat Liver Microsomes

Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view. 1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones. 2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours). 3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins. The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles.     

Paradoxical stimulation by 1-methyl-3-isobutylxanthine of rat liver cyclic AMP phosphodiesterase activity

DNA newly synthesized in UV irradiated Escherichia coli B/r Hcr⁺ was 2 min pulse-labeled at various periods, then denatured and analysed by sucrose gradient centrifugation either in neutral or in alkaline conditions. Data indicate that in DNA of damaged cells alkali-labile sites are produced. In cells saturated with inducible proteins production of alkali-labile sites disappears in ～1 h. In the absence of inducible proteins production of alkali-labile sites continues.     

Subcellular fractionation of epiphyseal cartillage isolation of matrix vesicles and profiles of enzymes,phospholipids, calcium and phosphate

Epiphyseal cartilage was fractionated into subcellular components by non-enzymatic methods, and analyzed for activity of marker enzymes, for phospholipids, and for calcium and inorganic phosphate. Alkaline phosphatase, a marker enzyme for matrix vesicles and plasma membranes, was concentrated in the 100 000 × g (microsomal) pellet and, upon subsequent frationalism, in the low-density fractions from the sucrose gradient. Mitochondrial and endoplasmic reticular enzymes were localized primarily in the 20 000 × g pellet, lysosomal enzymes predominantly in the supernate from the microsomal pellet. Two phospholipids characteristic of matrix vesicles, sphingomyelin and phosphatidylserine, were enriched in the low-density sucrose fractions; however, unlike matrix vesicles, there was no depletion in phosphatidylcholine or increase in lysophospholipids. Ca and inorganic P were concentrated in the higher-density fractions, the amounts in the lower-density fractions being some- what lower than those seen in matrix vesicles. The alkaline phosphatase-rich, low-density fractions were thus not identical to matrix vesicles isolated by collagenase digestion, but rather appear to be composed primarily of plasma membranes. Enzyme profiles indicate they were relatively free of mitochondrial, endoplasmic reticular and lysosomal contaminants. The data further indicate that significant modification of the phospholipid, electrolyte, and possibly enzyme content of chondrocyte plasma membranes, must occur during blebbing and matrix vesicle formation.     

A comparison of the subcellular distribution of 5′-nucleotidase, (Na+K+)-ATPase and adenyl cyclase in beef thyroid gland

The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na⁺K⁺)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na⁺K⁺)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N₂), 30 000 (M₂) and 165 000 × g (P₂) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na⁺K⁺)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N₂ and M₂. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M₂ and P₂. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid.     

Presence de glycosyltransferases a accepteurs chromatiniens dans les membranes nucleaires d'hepatocytes de singeThe presence of glycosyltransferases on chromatin acceptors in monkey liver nuclear membranes

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100 000 × g. The yield was 2.2 · 10⁷ nuclei per g of liver, and 70% of the homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively.The nuclei are hydrolysed by DNAase I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100 000 × g, under a buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.     

Protein kinases in brown adipose tissue of developing rats II. Two soluble kinase activities and their affinities for nucleotide and protein substrates

A heat-stable, soluble component of brown adipose tissue from newborn rats was found to be readily phosphorylated by protein kinase of the same subcellular fraction. The concentration of this component in brown fat decreased with the age of the animals. A boiled crude microsomal preparation from rat liver was also phosphorylated by brown fat protein kinase. The GTP-linked phosphorylation of the endogenous heat-stable protein was not stimulated by ATP (in contrast to phosphorylation of histone). The maximum velocity of phosphorylation achieved with GTP was about 2.5 times higher than that with ATP as nucleotide substrate. This difference was not due to ATPase activity in the assay. With histone as the protein acceptor both activities were the same. The affinity of protein kinase(s) for ATP was lower with the endogenous heat-stable brown-fat protein and with boiled microsomes (K_m of 0.21 mM and 0.17 mM, respectively) than with histone (K_m of 0.05 M). No detecable ATPase activity was present in either acceptor protein. It is concluded that the 100 000 × g supernatant fraction from brown fat of infant rats contains two protein kinase activities. One preferentially uses ATP and histone as substrates and the other uses endogenous heat-stable soluble proteins and either ATP or GTP.     

Occurrence of adenosine 3′:5′-cyclic monophosphate in plant tissues

A procedure is described which unequivocally demonstrates the presence of adenosine 3′:5′-cyclic monophosphate in Phaseolus vulgaris. Its concentration was determined spectrophotometrically at 2·6–9·2 nmol g^?1 of tissue (dry wt) for 6-day-old seedlings and about one-tenth of this in 13-day-old plants.     

MECHANISM OF THE CATION EFFECT IN SUBFRACTIONATION OF MICROSOMES

It was previously found that cations introduced into a discontinuous sucrose gradient exert a very pronounced effect on microsomal vesicles, and this principle proved to be effective in microsomal subfractionation. The mechanism of the cation effect was investigated. By using the radioactive isotopes ¹³⁷Cs and ⁸⁵Sr, it could be calculated that the amount of ions bound to the various subfractions increases their density by 0.14%, thereby enhancing the sedimentation velocity by only ~7%. In the presence of Cs⁺ the total volume of the microsomal pellet was decreased by ~15%. Assuming this change in volume to be due to a contraction of the individual vesicles, a roughly 2½-fold increase in sedimentation velocity would be expected. It is further demonstrated, on the basis of light scattering and millipore filtration experiments, that monovalent cations cause an extensive aggregation of rough microsomes and a less pronounced aggregation of smooth microsomes. The mean radius of the sedimenting particles of rough microsomes was found to be at least doubled or trebled in the presence of Cs⁺, which would give a 4- to 9-fold increase in the sedimentation velocity. Aggregation, therefore, appears to be the main factor in the accelerated sedimentation of rough microsomes in the presence of CsCl. Divalent cations exert a similar effect on a subfraction of the smooth microsomes. Isolated smooth microsomes are very unstable and often exhibit spontaneous aggregation. The presence of attached ribosomes, however, appears to impart greater stability to the rough microsomes as well as increasing their ability to bind monovalent cations. The primary cause of the aggregation of microsomal vesicles is probably due to a change in net charge.     

Fluctuations du taux d'AMP cyclique et des activites adenylate cyclase et AMP cyclique-phosphodiesterase dans les cultures synchrones d'un procaryote, Nocardia restricta

Cultures of Nocardia restricta, a prokaryote from the group of Actinomycetes, can be synchronised by diluting, in a fresh growth medium, cells already in stationary phase. The synchronisation of the cultures is monitored by examining the synchrony of DNA replication.In these synchronised cultures, the intracellular cyclic AMP level exhibits rythmic oscillations with a period equal to the generation time of the culture. There is only one peak per generation. The average ratio of maximum to minimum concentrations is at least 3.Cyclic AMP accumulates also in the medium with a step pattern. It appears in the medium during maximum production of cyclic AMP in the cell.The specific activity of adenylate cyclase (EC 4.6.1.1) measured in the 30 000 × g pellet of cell-free extracts also oscillates and correlates well with fluctuations in the cyclic AMP level. At the end of exponential growth, cyclic-AMP phosphodiesterase (EC 3.1.4.17) is detectable in the cells. The specific activity of this enzyme measured in the 30 000 × g supernatant of cell-free extracts shows also an oscillating pattern.To our knowledge it is the first time that such oscillations in the metabolism of cyclic AMP are described among prokaryotes. It is now possible to look at a link between this phenomenon and the cell cycle of the organism.     

SUBFRACTIONATION OF SMOOTH MICROSOMES FROM RAT LIVER

Total smooth microsomes from rat liver isolated on a Cs⁺-containing sucrose gradient were concentrated and subsequently fractionated by zone centrifugation on a stabilizing sucrose gradient. The prerequisite for fractionation is to prepare total smooth microsomes in a nonaggregated condition, as well as to utilize a procedure which counteracts enzyme inactivation. The median equilibrium density of the various smooth microsomal vesicles ranges from 1.10 to 1.18. The phospholipid/protein ratio is identical in all subfractions, but cholesterol, on a PLP basis, is enriched in the subfractions with the highest sedimentation velocity. The enzyme distribution pattern reveals a pronounced heterogeneity. A number of NADH- and NADPH-oxidizing enzymes are concentrated in the upper part of the gradient and exhibit a certain degree of separation from G6Pase. Mg⁺⁺-ATPase and AMPase are enriched in the lower part of the gradient. No specific enrichment of newly synthesized NADPH-cytochrome c reductase activity occurs in any of the subfractions after phenobarbital treatment. These data demonstrate that smooth microsomes, by adequate fractionation procedure, can be separated into subfractious of heterogeneous composition.     

PERMEABILITY OF MICROSOMAL MEMBRANES ISOLATED FROM RAT LIVER

Water compartments, permeability, and the possible active translocation of various substances in rat liver microsomes were studied by using radioactive compounds and ultracentrifugation. The total water of the microsomal pellet, 3.4 µl/mg dry weight, is the sum of water in the extramicrosomal and intramicrosomal spaces, or 56 and 44%, respectively. Sucrose space accounts for 77% of the intramicrosomal water and the hydration water ~ 14%, leaving almost no sucrose-impermeable space when using the ultracentrifugation approach. With increasing sucrose concentration, microsomes do not show an osmotic response. The intramicrosomal water decreases greatly in the presence of Cs⁺ and Mg⁺⁺ in rough but not in smooth microsomes. Uncharged substances of molecular weight of up to at least 600 freely penetrate microsomal membranes, which already become impermeable to charged substances at a molecular weight of 90. These substances also induce an osmotic response. The vesicles can be made permeable to charged substances after water treatment and cooling, which, however, does not increase glucose-6-phosphatase and inosine diphosphatase (IDPase) activities, and these enzymes can still be activated by deoxycholate. IDPase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation reactions, performed in vitro, also disproved the hypothesis of an accumulation of charged substances inside of vesicles of being a major pathway. The products of the enzymic reactions as well as the glucuronidated form of a hydroxylated product can be recovered on the cytoplasmic side of membranes, and little accumulation occurs in the intravesicular compartment.     

Effects of chymotrypsin and a calcium-dependent protein activator on guanosine 3′,5′-monophosphate phosphodiesterase from rat liver

Cyclic GMP phosphodiesterases from 100 00 × g rat liver supernatant were partially resolved by chromatography on DEAE-cellulose. Multiple forms of cyclic GMP phosphodiesterase(s) that were activated to different degrees by calcium plus a low molecular weight protein from rat liver and bovine brain supernantants, or by limited exposure to chymotrypsin, were identified. The cyclic GMP phosphodiesterase in some column fractions was activated over 10-fold by calcium plus activator or chymotrypsin. Activation by chymotrypsin was dependent both on the time of incubation with protease and its concentration. Prolonged exposure to chymotrypsin resulted in a decrease in s_20,w by sucrose density gradient centrifugation. The chymotrypsin-treated enzyme was no longer activated by exposure to calcium plus activator. The calcium- and protein activator-stimulated enzyme was inactivated by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA). Exposure of this activated enzyme to chymotrypsin did not result in further activation, but the chymotrypsin-treated enzyme was no longer inhibited by EGTA. The apparently irreversible effects of chymotrypsin and the reversible effects of calcium plus activator on cyclic GMP hydrolysis by the phosphodiesterase over a wide range of cyclic GMP concentrations appeared to be identical.     

Isomaltulose production from sucrose by Protaminobacter rubrum immobilized in calcium alginate

Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y_x/s of 0.295 g of cells/g sucrose and a specific growth rate (μ) of 0.192 h⁻¹. The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 °C an isomaltulose yield of 89–94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 °C ranged from 1.6 to 4.0 g isomaltulose g⁻¹ pellet h⁻¹, respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations.     

Factors affecting the adrenocorticotropic hormone stimulation of rabbit adrenal 17α-hydroxylase activity

Adrenocorticotropic hormone (ACTH)-stimulated 17α-hydroxylase activity of rabbit adrenal tissue has been shown to be associated with the subcellular fractions sedimented from 0.25 M sucrose at 33 000 × g for 60 min and at 105 000 × g for 60 min. The fraction sedimenting at 9000 × g for 20 min (mitochondria) contained the majority of the 11β-hydroxylase activity but also had a significant amount of 17α-hydroxylase activity. All subcellular 17α-hydroxylase activity showed an apparent preference for pregnenolone over progesterone. A 1 : 1 mixture of wholehomogenates of adrenal tissue from control and ACTH-stimulated rabbits incubated with[4-¹⁴C]pregnenolone synthesized as much 17α-hydroxylated corticosteroids as homogenate from the ACTH-stimulated tissue alone. However, the mixed homogenate synthesized only 1/4th–1/5th as much 17-deoxycorticosteroids as control, non-stimulated tissue, suggesting that the control tissue contained no inhibitor of 17α-hydroxylation, whereas ACTH-stimulated tissue may contain an inhibitor of 17-deoxycorticoid formation. 24-h dialysis of whole homogenates and subcellular fractions of adrenal tissue from control and ACTH-stimulated animals showed that 17α-hydroxylation was not activated in control tissue and somewhat inactivated in ACTH-stimulated tissue by this treatment. On the other hand, dialysis activated 17-deoxycorticoid formation by whole homogenates, but not in subcellular fractions, of both ACTH-stimulated and control adrenal tissue. Injection of 5 mg/kg cycloheximide prior to the first of 2 daily ACTH injections caused an average of 270 g body weight loss while not affecting the increase in adrenal weight effected by the ACTH. Adrenal tissue homogenates from cycloheximide injected animals produced only 50% as much 17α-hydroxycorticosteroids as homogenates of tissue from animals injected with ACTH alone and produced an amount of17-deoxycorticoids intermediate between homogenates of control and ACTH-stimulated tissue, suggesting the requirement of protein synthesis for 17α-hydroxylation stimulating activity of ACTH.     

Insulin degradation by human skeletal muscle

Although previous studies from this and other laboratories have extensively characterized insulin degrading activity in animal tissues, little information has been available on insulin responsive human tissues. The present study describes the insulin degrading activity in skeletal muscle from normal human subjects. Fractionation of a sucrose homogenate of skeletal muscle demonstrated that 97% of the total neutral insulin degrading activity was in the 100 000 × g supernatant with no detectable glutathione-insulin transhydrogenase activity. The 100 000×g pellet contained 85% of the total acid protease activity and all the glutathione-insulin transhydrogenase activity. The soluble insulin degrading activity was purified 1400-fold by ammonium sulfate fractionation, molecular exclusion, ion-exchange and affinity chromatography. Enzymatic activity was determined by measuring an increase in trichloroacetic acid-soluble products of the ¹²⁵I-labeled hormone substrates. The purified enzyme showed marked proteolytic specificity for insulin with a K_m of 1.63·10^?7 M (±0.32) and was competitively inhibited by proinsulin and glucagon with K_i values of 2.1 · 10?⁶ M and 4.0 · 10^?6 M, respectively. This insulin protease exhibited a pH optimum between 7 and 8, a molecular weight of 120 000 and was capable of degrading glucagon. Inhibition studies demonstrated that a sulfhydryl group is essential for activity. Molecular exclusion chromatography of [¹²⁵I]insulin degraded products revealed a time-dependent increase in degradation products with molecular weights intermediate between intact insulin and iodotyrosine. These studies demonstrate that the major enzymatic system responsible for insulin degrading activity is a soluble cysteine protease capable of rapidly metabolizing insulin under physiologic conditions.     

sn-Glycerol-3-phosphate transacylase activity in Euglena gracilis organelles

sn-Glycerol-3-phosphate transacylase activity was demonstrated in Euglena mitochondria, chloroplasts, and microsomes. There was no activity in the 100,000g 1-h supernatant. Exposure of each of the isolated organelles to 1 × 10^?4% Triton X-100 resulted in release of substantial quantities of transacylase activity into the 100,000g supernatant. Products formed by catalysis by the membrane-bound transacylases were heterogenous, while those resulting from catalysis by the extracted enzymes were practically all lysophosphatidate.