Transmembrane electrical potential affects the lipid composition of Acholeplasma laidlawii

In membranes of Acholeplasma laidlawii, lipid composition is regulated as a function of several stimuli affecting the volume and length of the hydrocarbon chains and the hydrocarbon-water interfacial area. This regulation is vizualized as changes in the relative amounts of the major polar lipids monoglucosyl diglyceride and diglucosyl diglyceride. These lipids form reversed hexagonal and lamellar phases with water, respectively. However, mixtures of the two lipids, in the molar proportions found in the A. laidlawii membrane, form a lamellar phase. By adjustment of the glycolipid ratio as a response to environmental stimuli, a certain stability of the lamellar membrane is maintained. In growing cells with oleoyl membrane lipids, a transmembrane electrical potential of approximately -50 mV (inside negative), but no transmembrane pH difference, was found. Addition of the K+ ionophore valinomycin caused a rapid and dose-dependent hyperpolarization remaining for at least 7 h. Simultaneously, a rapid and lasting metabolic decrease in the ratio monoglucosyl diglyceride/diglucosyl diglyceride occurred. The increase in potential and the decrease in the lipid ratio were both reversed in a dose-dependent manner by extracellular KCl. Likewise, the lipophilic cation tetraphenylphosphonium caused a dose-dependent decrease in membrane potential and an increase in the monoglucosyl diglyceride/diglucosyl diglyceride ratio, respectively. The ionophores monensin and particularly nigericin had similar but less pronounced effects on the potential and lipid ratios as valinomycin. The uncoupler carbonyl cyanide m-chlorophenylhydrazone had no effect on cell growth, membrane potential, or lipid regulation at 10 microM. These dissimilar structures and the low concentrations used make a direct disturbance of drug molecules on lipid packing in membranes less likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of Glucosyl Diglycerides by Mycoplasma laidlawii Strain B

Monoglucosyl diglyceride is synthesized from 1,2-diglyceride and uridine-5'-diphosphoglucose (UDP); diglucosyl diglyceride from monoglucosyl diglyceride, and uridine-5'-diphosphoglucose by membranes of Mycoplasma laidlawii strain B. All of these enzymatic activities reside in the membrane. Membranes solubilized by detergent action or succinylation and acetone powders of membranes were inactive. Requirements for Mg(2+), UDP, and appropriate lipid acceptor were demonstrated for biosynthesis of both glycolipids. Glucose-1-phosphate plus uridine triphosphate could replace the UDP requirement. A medium of relatively high ionic strength and a critical concentration of sodium lauryl sulfate stimulated biosynthesis of the monoglucosyl diglyceride. The optimal pH for both reactions was 8.0. A specificity for 1,2-diglyceride from the homologous organism was found for optimal synthesis of the monoglucosyl diglyceride, and a specificity for monoglucosyl diglyceride was found in the case of diglucosyl diglyceride synthesis. Both reactions were specific for UDP.     

Fatty Acid Composition of the Complex Lipids of Staphylococcus aureus During the Formation of the Membrane-bound Electron Transport System

In Staphylococcus aureus, 64 fatty acids could be separated by gas-liquid chromatography. The fatty acids consisted of normal, iso, and anteiso saturated fatty acids of from 10 to 21 carbon atoms. Of the total fatty acids, 2 to 4% were normal, iso, and anteiso monoenoic fatty acids. Positional isomers of the normal monoenoic fatty acids could be detected. The fatty acids could be extracted, leaving 1 to 2% of the total fatty acids in the residue. The proportions of the fatty acids in the residue and the total lipids differed significantly. The lipid extract contained less than 0.12% free fatty acid. Between 5 and 10% of the lipid fatty acids were associated with neutral lipids. The majority of the fatty acids were associated with the complex lipids: mono- and diglucosyl diglyceride, phosphatidyl glycerol, lysyl phosphatidyl glycerol, and cardiolipin. The proportions of the fatty acids changed markedly between bacteria grown anaerobically (no membrane-bound electron transport system) and those grown aerobically (containing a functional electron transport system). In each of the complex lipids, the proportions of the fatty acids, as well as the magnitude and direction of change in the molar quantity of the fatty acids per bacterium, changed dramatically between these growth conditions. Since the glucosyl diglycerides and phospholipids were formed from the same pool of diglyceride intermediates, the marked differences in fatty acids indicate that acyl transferase activities must be an important part of complex lipid metabolism in S. aureus.     

Lipid composition of Mycoplasma neurolyticum

The total lipid content of Mycoplasma neurolyticum comprises about 14% of the dry weight of the organisms and is about equally distributed between the phospholipid and the neutral-glycolipid fractions. The neutral lipids were identified as triglycerides, diglycerides, and cholesterol. The glycolipid fraction contained 1-O-beta-glucopyranosyl-d-2,3-diglyceride and 1-[O-beta-d-glycopyranosyl-(1-->6)-O-beta-d-glucopyranosyl]-d-2,3-diglyceride. The latter lipid is structurally identical to the diglucosyl diglyceride which occurs in Staphylococcus aureus. The phospholipids of the organism consist of a fully acylated glycerophosphoryl-glycerophosphoryl glycerol, phosphatidic acid, diphosphatidyl glycerol, phosphatidyl glycerol, and amino acyl esters of phosphatidyl glycerol. Phosphatidic acid and phosphatidyl glycerol account for greater than 90% of the phospholipids of organisms in the exponential phase of growth. The predominant fatty acids found in all of the acyl lipids were palmitic, stearic, and oleic acids.     

Heptose-containing pentaglycosyl diglyceride among the lipids of Acholeplasma modicum

A pentaglycosyl diglyceride with the tentative structure of galactosyl-galactosyl-mannoheptosyl-glucosyl-glucosyl diglyceride was found to be the major glycolipid in Acholeplasma modicum. The heptose is d-glycero-d-mannoheputose. The diglyceride-terminating moiety possesses the structure O-alpha-d-glucopyranosyl-(1 --> 2)-O-alpha-d-glucopyranosyl-sn-1,2-diglyceride. Other glycolipids occurring in this organism are a diglucosyl diglyceride and a monoglucosyl diglyceride with structures identical to the terminal segments of the pentaglycosyl diglyceride. More fully acylated derivatives of these two glycolipids also occur. The phospholipids are all of the glycerophosphoryl type. The neutral lipids are composed of diglycerides and four polyterpenes. The polyterpenes consist of both colored and colorless carotenoids and become radiolabeled with both [(14)C]acetate and [(14)C]mevalonate.     

The chemical composition of the membranes of protoplasts and L-forms of Staphylococcus

Membrane fractions were prepared from Staphylococcus aureus H and 100 after dissolution of the cell walls by a lytic enzyme from Streptomyces griseus. Membranes were also prepared from the L-forms derived from the same strains. The membranes were analysed for protein, lipid, carbohydrate and RNA contents, and the fatty acid composition of the lipids was determined. A branched-chain saturated C(15) acid was the major component in all samples, and the correspondence between L-forms and parent bacteria was fairly close. The lipids were separated into non-polar-lipid, glycolipid and phospholipid fractions; the L-forms contained a little more neutral lipid and much more glycolipid than the parent bacteria. In all membranes the glycolipid, which accounted for all the carbohydrate present, was a diglucosyl diglyceride. The major phospholipids of the protoplast membranes were phosphatidylglycerol and some lipoamino acids (lysine and a little alanine). On the other hand, diphosphatidylglycerol was the chief phospholipid found in L-form membranes.     

Lipoteichoic Acid Localization in Mesosomal Vesicles of Staphylococcus aureus

Mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P have been prepared and examined for the presence of lipoteichoic acid. Lipids were first removed by treatment with pyridine-acetic acid-butanol (22:31:100, vol/vol/vol) and chloroform-methanol (2:1, vol/vol). Subsequently, lipoteichoic acid was removed with 40% phenol in water. The lipoteichoic acid from mesosomal vesicles was characterized by (i) equimolar glycerol and phosphate, (ii) alanine upon hydrolysis (2 N NH₄OH, 18 h, 22 C), and (iii) fatty acids, diglycerol triphosphate, glycerol monophosphate, and glycerol diphosphate upon alkaline hydrolysis (1 N NaOH, 3h, 100 C). The plasma membranes contained no lipoteichoic acid. The presence in mesosomal vesicles of 18% of the dry weight as lipoteichoic acid and its absence from plasma membranes provide the first major chemical differences between these organelles. A study of the lipoteichoic acid content in various fractions of the cell showed that the mesosomal vesicles were the major and probably the sole site for the localization of the lipoteichoic acid in these organisms. A new method for the preparation of mesosomes in increased yields is reported. A theory for the control of cell division involving lipoteichoic acid and the mesosome is proposed.     

MEMBRANE SYNTHESIS IN BACILLUS SUBTILIS : III. The Morphological Localization of the Sites of Membrane Synthesis

The results of several lines of investigation indicate that membrane growth in Bacillus subtilis does not occur at one or a small number of discrete zones. No indications of large regions of membrane conservation were observed. Kinetic labeling experiments of mesosomal and plasma membrane lipids indicate that the mesosomal lipids are not precursors of the plasma membrane lipids. Density shift experiments, in which the changes in buoyant density of membranes were studied after growth in deuterated media, showed no indication of large zones of conservation during membrane growth. Radioautography of thin sections of cells pulse labeled with tritiated glycerol showed no indication of specific zones of lipid synthesis. The consequences of these results for models of cell growth and division are discussed.     

Isolation and properties of mesosomal membrane fractions from Micrococcus lysodeikticus

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg(2+). 5. Mesosomal membrane vesicle fractions prepared at differing Mg(2+) concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b(556) was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome.     

Extraction, Characterization, and Cellular Localization of the Lipids of Staphylococcus aureus

Satisfactory extraction and assay procedures have been developed for the lipids of Staphylococcus aureus. The following lipids have been characterized in detail: the vitamin K(2), which is shown to exist as isoprenologues with side chains of 35, 40, and 45 carbon atoms; monoglucosyldiglyceride and diglucosyldiglyceride, which account for all the carbohydrate in the lipid extracts; the lysyl ester of phosphatidyl glycerol, phosphatidyl glycerol, and cardiolipin, which account for 98% of the phosphate in the lipid extract. The extraction procedure removes 98% of the total bacterial fatty acids. Acidification of the medium before harvest and refluxing in isopropanol are critical in the extraction procedure for the maximal recovery of lysyl-phosphatidyl glycerol and the glucolipids. The lipids have been shown to be a part of the same membrane as the respiratory pigments.     

The lipid composition of a halotolerant species of Staphylococcus epidermidis.

Studies were carried out on the lipid composition of a halotolerant Staphylococcus epidermidis isolated in pure culture from a growth medium for extreme halophiles containing 25% NaCl. The four major polar lipid components in this bacterium were found to be: (a) glycerophosphoryl diglucosyl diglyceride (10% by weight) with structure 3(1)-O-(-sn-glycerol-1-phosphoryl-6'-O=(beta-D glucopyranosyl-(1 leads to 6)- O-beta-D-glucopyranosyl)-1(3),2-diacyl-sn-glycerol; (b) diglucosyl diglyceride (15% by weight) with structure 3(1)-O-(beta-D-glucopyranosyl (1 leads to 6)-O-beta-D-glucopyranosyl)-1(3),2-diacyl-sn-glycerol; (c) monoglucosyl diglyceride (3% by weight) with structure 3(1)-O-(beta-D-glucopyranosyl)-1(3),2-diacyl-sn-glycerol, and (d) phosphatidylglycerol (60% by weight) with structure 1,2 diacyl-sn-glycero-3-phosphoryl-1'-sn-glycerol. Phosphatidic acid, cardiolipin, lysophosphatidylglycerol and three unidentified phospholipids were also detected in small amounts. Each lipid component had essentially the same fatty acid composition namely, anteiso-15:0 (60-75%), anteiso-17:0 (18-24%), iso-17:0 (8--10%), and small amounts of palmitic and stearic acids (2-5%). The fatty acids were non-randomly distributed in phosphatidylglycerol, the shorter chain anteiso 15:0 fatty acid being exclusively esterified to the 2-position and the longer chain anteiso- and iso-17:0 fatty acids at the 1-position. The fatty acid composition was not affected by increaseing NaCl content in the medium in the rande 0--15% but the proportion of anteiso-15:0 increased greatly when the salt concentration was increased to 25%. The proportions of ionic polar lipids were modified to give an increased net negative charge per mol ionic lipids when NaCl in the medium was increased from 15 to 25%, but the proportions of neutral glycolipids remained fairly constant.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus.

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated. Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [14C]mannose from GDP-[14C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [14C]mannosyl-1-phosphorylundecaprenol. In contrast, this is the major mannolipid synthesized from GDP-[14C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents. Both membrane preparations possessed the ability to catalyse the transfer of [14C]mannose from purified [14C]mannosyl-1-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [14C]mannosyl units from 14C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

The Separation and Isolation of Plasma Membranes and Mesosomal Vesicles from Staphylococcus Aureus

We have separated and Isolated the plasma membranes and mesosomal vesicles of Staphylococcus aureus ATCC 6538P. Cells were grown aerobically in Difco synthetic AOAC broth, washed and resuspended in hypertonic buffer (3.45 M NaC1) containing 0.02 M MgSO₄. Cell wall was removed by treatment with lytic enzyme from S. aureus, strain LS. The protoplasts were collected by centrifugation at 10,000 × g for 1 hour, resuspended in hypotonic buffer containing 0.02 M MgSO₄ and lysed. The resultant plasma membranes were washed and centrifuged on a 60tr>75Z sucrose density gradient at 55,000 × g for 15 hours. Gradient patterns showed two bands of membranes. Crude mesosomes were obtained from the 10,000 × g supernatant fractions by centrifugation at 100,000 × g for 2 hours. The reddish-brown gelatinous pellet, which consisted of mesosomal vesicles and a few ribosomes, was washed and centrifuged on a 60 to 85% sucrose density gradient at 100,000 × g for 15 hours. Gradient patterns produced two bands of mesosomal vesicles. The homogeneity of the plasma membranes and mesosomal vesicles was determined by electron microscopy and chemical analyses.     

Diglyceride Kinase Mutants of Escherichia coli: Inner Membrane Association of 1,2-Diglyceride and Its Relation to Synthesis of Membrane-Derived Oligosaccharides

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.     

Preparation and chemical properties of the outer membrane of a moderately halophilic gram-negative bacterium.

Outer membranes, almost free from peptidoglycan components, were prepared from a moderately halophilic gram-negative bacterium grown in a medium containing 2 M NaCl. The outer membrane was easily released, leaving mureinoplasts, by mild desalting in a 20% sucrose solution containing 50 mM tris(hydroxymethyl)aminomethane-HCl buffer, pH 7.8. The membrane was recovered by treatment with DNase I and CsCl buoyant density centrifugation. Chemical analyses revealed that the outer membrane was mainly composed of 31% protein, about 20% extractable lipids (mainly phospholipids), and lipopolysaccharides. The proteins had about 18 mol % excess of acidic over basic amino acids. The phospholipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, cardiolipin, and an unidentified phospholipid containing glucose, which seemed mainly associated with the outer membrane. The content of lipopolysaccharides in the outer membrane was calculated arbitrarily as 30% from the heptose content. A unique feature of these lipopolysaccharides seemed to be higher lipid content than found in lipopolysaccharides of other gram-negative bacteria. The major fatty acids of bound lipids of the outer membrane resembled those of the lipopolysaccharides obtained from cell envelope preparation and contained high concentrations of 3-hydroxy lauric acid.     

Inhibition of wall autolysis in Streptococcus faecalis by lipoteichoic acid and lipids.

Fully acylated lipoteichoic acid (LTA) isolated from Streptococcus faecalis ATCC9790 (S. faecium) inhibited autolysis of walls from the same organism at concentrations (1.0 to 1.5 nmol of LTA per mg of wall) comparable to those found in intact cells. Partially deacylated LTA isolated from S. faecalis or chemically deacylated LTA failed to inhibit significantly in the same concentration range. Beef heart cardiolipin and commercially obtained dipalmitoyl phosphatidyl glycerol were also found to inhibit wall autolysis in S. faecalis. Chemical deacylation of beef heart cardiolipin also removed the inhibitory activity of this molecule. Lipid fractions isolated from S. faecalis that inhibited wall autolysis were: diphosphatidyl glycerol (cardiolipin), phosphatidyl glycerol, aminoacyl phosphatidyl glycerol, and a neutral lipid fraction. Glycolipids were not found to be effective inhibitors. The possible role of LTA and/or certain lipids as regulators of cellular autolytic activity is discussed.     

Distribution of enzymes involved in mannan synthesis in plasma membranes and mesosomal vesicles of Micrococcus lysodeikticus

The distribution of membrane-bound enzymes involved in mannan biosynthesis in plasma and mesosomal membranes of Micrococcus lysodeikticus has been investigated.Isolated mesosomal vesicles, unlike plasma membrane preparations, cannot catalyze the transfer of [¹⁴C]mannose from GDP-[¹⁴C]mannose into mannan. This appears to result from the inability of this membrane system to synthesize the carrier lipid [¹⁴C]mannosyl-l-phosphorylundecaprenol. In contrast, this is the major manno-lipid synthesized from GDP-[¹⁴C]mannose by isolated plasma membranes. The possibility that substrate inaccessibility could account for the failure to detect the enzyme in isolated mesosomal vesicles appears unlikely from the lack of activity following disruption of the vesicles with ultrasound or with surface active agents.Both membrane preparations possessed the ability to catalyse the transfer of [¹⁴C]mannose from purified [¹⁴C]mannosyl-l-phosphorylundecaprenol into mannan. Furthermore, free mannan and mannan located on both unlabeled mesosomal and unlabeled plasma membranes could act as acceptors of [¹⁴C]mannosyl units from ¹⁴C-labeled carrier lipid located in prelabeled plasma membranes. The possibility that the juxtaposition of mesosomal vesicles and enveloping plasma membrane (i.e. the mesosomal sacculus) in vivo allows mannan, located on mesosomal vesicles, to accept mannosyl units from carrier lipid located in the sacculus membrane is discussed.     

Cellular localization of lipoteichoic acid in Streptococcus faecalis.

The release of lipoteichoic acid and mesosomal vesicles to the supernatant buffer during the formation of spherical, osmotically fragile bodies was studied using Streptococcus faecalis ATCC 9790. Autolytic N-acetylmuramidase action was permitted to take place in exponential-phase cells incubated in a buffer which provides an exceptional degree of osmotic stabilization. Both lipoteichoic acid and mesosomal vesicles were relatively rapidly released to the supernatant buffer. Most of the cellular content of lipoteichoic acid (and mesosomal vesicles) was found in the supernatant buffer at incubation times when the cells still retained over 75% of their cell wall. [14-C]- or [3-H]glycerol was used as a label for both cellular lipoteichoic acids and lipid-glycerol. Glycerol in lipoteichoic acid was quantitated after phenol-water and chloroform-methanol treatments and identified by products of acid hydrolysis and its ability to be precipitated by (i) antibodies specific for the polyglycerol-phosphate backbone, (ii) antibodies to the streptococcal group D antigen, and (iii) concanavalin A. Evidence was obtained that lipoteichoic acid was not associated with isolated mesosomal vesicles. Centrifugation of supernates at 200,000 X g sedimented membranous (mesosomal) vesicles and nearly all of the lipid-glycerol present, whereas essentially all of the lipoteichoic acid remained in the supernatant. The sedimented mesosomal vesicles differed from protoplast membrane in their higher lipid-phosphorus to protein ratio and in the absence of detectable levels of two enzymatic activities found in protoplast membranes, adenosine triphosphatase and polynucleotide phosphorylase. Both types of membranes were found to contain DD-carboxypeptidase and LD-transpeptidase activities at nearly the same specific activities. No evidence was obtained for the association of autolytic N-acetylmuramidase activity with either type of membrane preparation.     

Phospholipids and glycolipids of sterol-requiring Mycoplasma

The phospholipids of Mycoplasma hominis type 2 strain 07 are composed almost entirely of phosphatidyl glycerol. Traces of other glycerophospholipids may exist. No glycolipids are found. The phospholipids of Mycoplasma sp. avian strain J are composed of diphosphatidyl glycerol, which predominates in older cultures, a monoacyl glycerophosphoryl glycerophosphate, which may serve as a precursor of diphosphatidyl glycerol, and phosphatidyl glycerophosphate. This organism also contains cholesteryl glucoside and an unidentified glycolipid which appears to be similar to a monoglucosyl diglyceride. No turnover or radioisotope labeling of the phospholipids occurs during metabolism. This lack of turnover during growth is indicative of a structural role for these glycerophospholipids. A concomitant decrease of monoacyl glycerophosphoryl glycerophosphate and increase of diphosphatidyl glycerol occurs during growth.     

Negligible release of cardiolipin during milk secretion by the ruminant

The presence of cardiolipin (diphosphatidyl glycerol) in lactating mammary tissue (cow and goat) was investigated. The tissue was separated into subcellular fractions by sedimentation; the identities of the fractions were confirmed by electron microscopy. Polar lipids recovered from the fractions, the whole tissues, and milks were analyzed by two-dimensional thin-layer chromatography and the percentages of cardiolipin were determined. The phospholipids of whole mammary tissue from the cow and goat contain 3-5% cardiolipin which is concentrated largely, if not exclusively, in the mitochondria. Although milk may on occasion have up to 1% cardiolipin in its phospholipids, some normal milks contain less than 0.15%. Since tissue contains 20-30 times the amount (mg/g) of phospholipids in milk, the quantitative ratio of tissue to milk cardiolipin is several hundred to one. We interpret this to mean that the mechanism of milk secretion is highly selective and insures retention of mitochondria within the cell even though they are decidedly smaller than milk fat globules which are continuously secreted. Our findings substantiate the conception that there is very little disintegration of the cell or disruption of the plasma membrane during milk secretion. The fatty acids of cardiolipin from lactating mammary tissue of cow, goat, and pig are highly unsaturated; they contain 50% or more octadecadienoic acid.