Sulphatide synthesis in isolated oligodendroglial and neuronal cells

—Cerebroside sulphotransferase activity in oligodendroglia from calf brain is 8-fold greater per cell than in calf neurons isolated at the same time under similar conditions. However, neuronal cell fractions from calf or rat brain have significant sulphotransferase activity, and in neurons isolated from rat brain at various ages, the capacity to synthesize sulphatide increases during myelination. The neuronal and oligodendroglial enzymes have similar substrate specificities and pH optima. Less of the enzyme could be extracted with Triton X-100 from the isolated cells than from microsomes prepared from whole brain.     

28 kDa adenosine-binding proteins of brain and other tissues.

Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the presence of 0.1% sodium cholate. Amino acid compositions of the 28 kDa proteins were similar, but not identical.     

ISOLATION AND CHARACTERIZATION OF A SOLUBLE GLUCOSE-CONTAINING SIALOGLYCOPROTEIN FROM THE CORTICAL GREY MATTER OF CALF BRAIN

A sialoglycoprotein has been isolated from the cortical grey matter of calf brain after homogenization in 0.32 M-sucrose or in 0.15 M-NaCl. The sialoglycoprotein is present in the supernatant obtained after centrifugation at 100,000 g for 60 min. It is designated GP-350 on account of its elution with 350 mM-NaCl on a DEAE-cellulose column. From DEAE-cellulose chromatography it is evident that compounds comparable to GP-350 occur in the brain of calf and sheep, whereas they seem to be absent in calf liver and kidney. After purification, with polyacrylamide gel electrophoresis only one band can be shown both at pH 8.9 and 7.5. GP-350 consists of about 83 percent of protein and about 17 per cent of carbohydrate. The polypeptide core has an acidic character: amino acid analysis gives 26 per cent for glutamic acid plus aspartic acid and their amides, with a ratio of acidic to basic amino acids of 3.3. The carbohydrate moiety contains 2.4% sialic acid, 5.5 % hexosamine and 9.4% hexose. It is remarkable that this brain sialoglycoprotein comprises 4% glucose. Care was taken to prevent contamination with glucose-containing materials during the purification procedure of GP-350. The complete absence of other glucose-containing compounds which occur in brain, Le. glycogen and gangliosides, was demonstrated. GP-350 accounts for at least 3 per cent of the total saline-extractable protein and about 20 per cent of the total saline-extractable protein-bound sialic acid of the cortical grey matter of calf brain. These percentages correspond to 390 pg of protein and to 14 μg of sialic acid per g wet weight. GP-350 remains soluble when the pH is brought to 3.9 or when ethanol is added to 70 % (v/v).     

MEMBRANE-BOUND NEURAMINIDASE IN THE BRAIN OF DIFFERENT ANIMALS: BEHAVIOUR OF THE ENZYME ON ENDOGENOUS SIALO DERIVATIVES AND RATIONALE FOR ITS ASSAY

—The activity of brain membrane-bound neuraminidase on endogenous and exogenous substrates was comparatively studied in various animals (rat, chicken, rabbit, pig, calf and human). The maximum rate of hydrolysis of endogenous substrates by membrane-bound neuraminidase (using a crude preparation of the enzyme) was different in the various animals (from 0·05 to 0·73 units, referred to 1 mg protein) and was obtained under similar but not identical optimum conditions (pH from 4·1 to 5·1; requirement or not of Triton X-100). The maximum degree of hydrolysis of endogenous substates was also different (from 15 to 27 nmol released NeuNAc/mg protein) and was obtained within different incubation periods (from 2 to 18 h). It corresponded (in rabbit, calf, human brain only), or not, to the actual exhaustion of the endogenous substrates. The endogenous substrates were recognized as both gangliosides and sialoglycoproteins. The extent of hydrolysis of sialoglycoproteins varied from 1·5% in rabbit to 15·6% in chicken brain; the hydrolysis of gangliosides (ranging from 14·1% in pig to 53·7% in rabbit brain) reached only in some animals (rabbit, calf, human) the complete transformation of major oligosialogangliosides into the neuraminidase resistant monosialoganglioside GMI. Upon addition of exogenous substrates (sialyl-lactose, ganglioside GD1a, brain sialopeptides, ovine submaxillary mucin) the actual rate of liberation of total NeuNAc (from both endogenous and exogenous substrates) considerably exceeded, although at a different extent (depending on the animal and on the added substrate used) the rate of hydrolysis of sole endogenous substrates. The possibility of an accurate assay of brain membrane-bound neuraminidase in a crude enzyme preparation is evaluated and guidelines for the assay procedure suggested.     

Glutamyl-Taurine Is the Predominant Synaptic Taurine Peptide

Several taurine-containing peptides have been identified from trichloracetic acid extracts of synaptosomes and their subcellular vesicles prepared from calf brain. These peptides contain aspartic and glutamic acids, serine and taurine, and are often present in an N-acetylated form. The peptides were isolated as single spots by TLC. Glutamyl-taurine was found to be the predominant structure when analyzed by fast atom bombardment (FAB) mass spectrometry.     

THE ACTION OF CALF BRAIN SIALIDASE ON GANGLIOSIDES, SIALOGLYCOPROTEINS AND SIALOGLYCOPEPTIDES

Abstract— In agreement with other investigators it has been shown that endogenous as well as added gangliosides are a substrate for brain sialidase. The release of sialic acid was enhanced in the presence of Triton X-100; this might be due to the action of the detergent on the ganglioside micelles. The sialic acid release from endogenous gangliosides was observed over 48 h and compared with the effect of the sialidase on the endogenous glycoproteins. Though the hydrolysis of sialic acid from gangliosides is much faster in the first hours, after 48 h 40 per cent of the total bound sialic was released from both substrates at pH 4.0 and 37°C.
Sialoglycopeptides obtained from brain glycoproteins are also metabolized by the sialidase. No effect of Triton X-100 on this substrate has been observed. From sialoglycopeptides, fractions can be obtained by DEAE-Sephadex A-50 column chromatography with a sialic acid content from 8 to 26 per cent. The fractions with a high sialic acid content were about equally active towards brain sialidase as gangliosides. The results agree with the similar turnover rate observed for the carbohydrate chains from gangliosides and glycoproteins, but are in contrast to the observations of other investigators who have stated that glycoproteins are a poor substrate for brain sialidase. In our experiments bovine and ovine submaxillary mucins and sialyl-lactoses showed only slight activity compared to gangliosides and selected brain sialoglycopeptides.     

Uridine diphosphate 2-deoxyglucose: Chemical synthesis,enzymic oxidation and epimerization

The paper describes chemical synthesis of uridine diphosphate 2-deoxyglucose (UDPdGlc) through reaction of uridine 5′-phosphomorpholidate with 2-deoxy-α-d-glucopyranosyl phosphate. The prepared analog of uridine diphosphate glucose (UDPGlc) served as a substrate for calf liver UDPGlc dehydrogenase (EC 1.1.1.22), the reaction product was identified as nucleotide deoxyhexuronic acid derivative. The apparent K_m for UDPdGlc was found to be 60 times that of UDPGlc, and the relative V value for the analog was 0.09. The peculiar lag-period in reaction kinetics has been observed for the analog, and is presumably connected with the slow rate of the initial stages of the reaction. UDPdGlc was found to be quite an efficient substrate for UDPGlc 4-epimerases (EC 5.1.3.2) from yeast, calf liver and mung bean seedlings.     

Affinity-chromatography purification of alkaline phosphatase from calf intestine.

A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.     

Lack of excision of 4HAQO adducts from DNA by cell extracts that excise pyrimidine dimers

A substrate of DNA containing 4HAQO adducts, suitable for studies of excision repair, was prepared by reacting calf thymus DNA with [3H]monoacetyl-4HAQO. A crude HeLa cell extract was prepared by the method of Mortelmans et al (Proc. Natl. Acad. Sci. U.S.A. 73, 2757, 1976). The cell extract would specifically excise pyrimidine dimers from UV-irradiated DNA but would not release 4HAQO adducts in an acid soluble form. This result points to different initial steps in the excision repair process for these two forms of damage even though much of the repair mechanism is common to both.     

The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II

The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM).     

1-Piperideine as an In Vivo Precursor of the γ-Aminobutyric Acid Homologue 5-Aminopentanoic Acid

Intraperitoneal injection of the cyclic imine 1-piperideine in mice resulted in measurable quantities of 5-aminopentanoic acid in brain. 5-Aminopentanoic acid is a methylene homologue of gamma-aminobutyric acid (GABA) that is a weak GABA agonist. 5-Aminopentanoic acid formed in the periphery was ruled out as the source of brain 5-aminopentanoic acid based on the absence of detection in brain following injection of 100 mg/kg of 5-aminopentanoic acid. Deuterium-labeled 1-piperideine was prepared by exchange in deuterated phosphate buffer. Injection of [3.3-2H2]1-piperideine yielded [2.2-2H2]5-aminopentanoic acid in brain. The results are consistent with uptake of 1-piperideine into brain and oxidation of the precursor to 5-aminopentanoic acid. Inhibition of GABA catabolism by pretreatment with aminooxyacetic acid increased brain concentrations of 5-aminopentanoic acid formed from 1-piperideine, suggesting that 5-aminopentanoic acid is an in vivo substrate of 4-aminobutyrate:2-oxoglutarate aminotransferase.     

On the mechanism of a calcium-associated defect of oxidative phosphorylation in progessive muscular dystrophy

Partially purified elongation factor 1 preparations from calf brain, sheep brain, calf liver, and rabbit reticulocytes have been compared in their ability to interact with GTP and Phe-tRNA. A nitrocellulose filter assay has been used to study these interactions, and with all the EF1 preparations studied, evidence has been obtained for the formation of a Phe-tRNA·-EF1·-GTP complex. The ternary complex reacts with calf brain ribosomes in the presence of poly(U) resulting in a rapid hydrolysis of GTP and the binding of Phe-tRNA to the ribosome. Indirect evidence indicates that EF1·GDP is a product of this reaction. In the absence of poly(U) the intact complex reacts with the ribosomes without hydrolysis of GTP. The stability of the ternary complex was different with the various EF1 preparations, but the most stable complexes were prepared with calf brain EF1. Sephadex chromatography of the ternary complex shows that it contains a low molecular-weight species of the enzyme.     

The structure and amount of tubulin in cells and tissues.

The NH2-terminals and amino acid compositions of three carboxymethylcysteine-containing peptides produced from carboxymethylated calf brain tubulin by digestion with elastase have been determined. The 3H/14C ratio obtained when these peptides were generated from mixtures of [14C]carboxymethylated calf brain tubulin and the [3H]carboxymethylated proteins of mouse brain or 3T3 cells and subsequently purified indicated that both mouse brain and 3T3 cells contained protein giving rise to peptides homologous to these peptides. This indicates that the tubulins of calf brain, mouse brain, and 3T3 cells are homologous in the regions of amino acid sequence accounting for the peptides examined. From the 3H/14C ratio of the isolated peptides, 13.5% of the protein of mouse brain and 3.5% of the protein of 3T3 cells were estimated to be tubulin.     

Brain dolichyl pyrophosphate phosphatase. Solubilization, characterization, and differentiation from dolichyl monophosphate phosphatase activity

Dolichyl [beta-32P]pyrophosphate ([beta-32P]Dol-P-P) has been prepared chemically to study Dol-P-P phosphatase in calf brain. Calf brain microsomes catalyze the enzymatic release of 32Pi from exogenous [beta-32P]Dol-P-P by a bacitracin-sensitive reaction. [32P]Pyrophosphate was not detected with the water-soluble product even when 1 mM sodium pyrophosphate was added to impede pyrophosphatase activity. A substantial fraction of the Dol-P-P phosphatase activity can be solubilized by treating brain microsomes with 3% Triton X-100. The detergent extracts catalyze the enzymatic release of 32Pi from [beta-32P]Dol-P-P and the conversion of [14C]undecaprenyl pyrophosphate to [14C]undecaprenyl monophosphate. The solubilized Dol-P-P phosphatase activity: 1) is optimal at neutral pH; 2) is inhibited by Mn2+ and stimulated by EDTA; 3) exhibits an apparent Km = 20 microM for Dol-P-P; 4) is competitively inhibited by undecaprenyl pyrophosphate, and 5) is blocked by bacitracin. Solubilized Dol-P-P phosphatase activity differs from Dol-P phosphatase activity present in the same detergent extracts with respect to: 1) thermolability at 50 degrees C, 2) effect of 20 mM EDTA, and 3) sensitivity to phosphate and fluoride ions. These studies describe the chemical synthesis of [beta-32P]Dol-P-P for use in a convenient assay of Dol-P-P phosphatase activity. A procedure for the solubilization of Dol-P-P phosphatase activity from microsomes is presented, and an enzymological comparison indicates that Dol-P-P and Dol-P phosphatase are separate enzymes in calf brain.     

Effect of acute cyanide intoxication on the active transport of amino acids in brain slices

(1) Acute hypoxia was produced in adult rats by cyanide inhalation and the effect on the active transport of amino acids was studied in brain slices. (2) Initial and steady-state accumulation of amino acids and rates of amino acid exit were identical in brain slices from control and treated animals when a glucose-containing incubation medium was used. (3) When the incubation was carried out in a glucose-free incubation medium, the inhibition of initial and steady-state accumulation and the stimulation of amino acid exit observed in control slices were significantly reduced or abolished in slices from treated animals. (4) Tissue swelling, size of ‘inulin space’ and glucose consumption did not differ in the two groups of animals. (5) Also the respiration rate was identical in slices from control and treated animals incubated in the presence of glucose. In the absence of added substrate, brain slices from treated animals consumed 15-20 per cent more oxygen than control slices. (6) A possible correlation between the effects observed on amino acid transport and on respiration is suggested. The reasons why cyanide given in vivo or added in vitro have different effects on amino acid transport in brain slices are discussed.     

Relationship between amino acid transport and electron transport by membrane vesicles of Micrococcus denitrificans

Membrane vesicles were prepared from Micrococcus denitrificans by osmotic shock of lysozyme spheroplasts. These vesicles concentrated 4 amino acids via two systems; one for glycine-alanine and the other for asparagine-glutamine. Amino acid transport was coupled to the membrane-bound electron transport system and involved interactions of the primary dehydrogenases, cytochromes, cytochrome oxidase and oxygen. After transport the amino acids were recovered unchanged from the vesicles. The substrates of the membrane-bound electron transport system d-lactate, l-lactate, formate, succinate, NADH, glucose-6-phosphate and α-glycerolphosphate all stimulated transport at least 2-fold. Both oxygen and nitrate could serve as terminal electron acceptors with vesicles prepared from cells grown anaerobically with nitrate. Anaerobic transport in the presence of nitrate was not inhibited by cyanide but was inhibited by nitrite. A system stimulated by substrates of the electron transport system but independent of added terminal electron acceptors was found also in the vesicles prepared from anaerobically grown cells. Addition of one combination of two substrates for electron transport produced an amino acid uptake 12 to 15% greater than the sum of the rates for each substrate added singly. Additions of other combinations gave rates of transport less than the sum of the rates of each added alone. Both the dehydrogenase activities and the coupling of electron transport to amino acid uptake were modified by changing the growth conditions and differences between the effectiveness of each substrate for each of the two transport systems could be detected. The efficiency of the vesicles per protoheme, the prosthetic group of the membrane-bound cytochrome b, with d-lactate as substrate was 27% for glutamine and 6% for glycine of the rates of transport of these two amino acids in intact cells when driven by endogenous respiration. Assuming one amino acid transported per electron, the transport of glycine utilized 1% of the respiratory capacity with glucose-6-phosphate as substrate. The coupling to the electron transport with the other substrates was less efficient. It appeared that a small portion of the total capacity of the electron transport system was coupled to amino acid transport and the coupling to respiration, as well as the primary dehydrogenase activities and terminal cytochrome oxidase, were modified in response to the conditions of growth.     

Performance of calves fed on milk replacers containing fish protein hydrolysate

Milk substitutes for calves are based upon dried skim milk and added fats of vegetable or animal origin. Many attempts have been made to replace part of the skim milk in these feeds by cheaper sources of protein.A milk product coagulates in the calf's abomasum and has a high digestibility. Milk replacers based on fish protein hydrolysate (FPH) do not coagulate in the abomasum, but have a high apparent digestibility coefficient (0.92) and the amino acid balance is similar to that of skim milk.Calves were fed on milk replacers prepared from FPH made from various materials. In general, FPH prepared from white fish (cod, blue whiting, white fish offal) was satisfactory although calf performance until weaning was poorer than for calves fed on milk replacers with dried skim milk (SMP). Some ways are suggested by which the quality of the product (FPH) could be maintained. FPH preparations from fatty fish, like sprats and mackerel, were also used. An antioxidant was added in the preparation of sprats and mackerel because of the unsaturated nature of fish fats. Milk replacers containing sprats prepared without an antioxidant and mackerel (31% body fat) with antioxidant were unsatisfactory and further development work is required on FPH materials from fatty fish.FPH from white fish can be dried without any appreciable loss in calf performance. The daily gain to weaning was 0.25 and 0.29 kg per day for calves fed on the dried and undried material, respectively. On the basis of present knowledge it is suggested that FPH can replace one third of skim milk in milk replacers for early weaned calves; the proportion recommended may rapidly increase with further technical development of the product.     

Enzyme Immunoassay of the Myelin Basic Protein

Abstract: An enzyme immunoassay using a double-antibody solid-phase technique for myelin basic protein (MBP) has been developed. Antisera were prepared by immunizing rabbits with the purified MBP from chick brain. The conjugation of MBP with horseradish peroxidase was performed by the periodate oxidation method in triethanolamine-acetate buffer (pH 8.5). The sample, antiserum, and conjugate were incubated at 4°C for 16 h, after which the insoluble second antibody was added and the reaction mixture was incubated at 4°C for 3 h. The peroxidase activity of the insoluble conjugate was assayed fluorometrically with hydrogen peroxide and 3-( p -hydroxyphenyl)propionic acid as substrates. The method had an analytical range from 50 pg to 1 ng (from 2.3 × 10⁻¹⁵ to 4.5 × 10⁻¹⁴ mol). The within-assay coefficient of variation (CV) was between 4 and 11% and the between-assay CV for 200 and 400 pg of MBP was 5.5 and 7.1%, respectively. A weak cross-reactivity was observed between chick MBP and bovine MBP, while no reactivity was shown with calf thymus histone. The MBP content of the brain during development increased markedly from the 3rd embryonic week to the 3rd post-hatch week (from 0.01 to 2.4 mg/g of fresh tissue), and the adult level was 3.2 mg/g of fresh tissue.     

Renin substrate (angiotensinogen) preparations in the determination of prorenin and renin: evidence for extrarenal plasma prorenin and its renal "convertase"

Standard methods for determining prorenin-renin concentrations in plasma (PRC) and other tissues require the addition of exogenous renin substrate (angiotensinogen) to improve the kinetics of the renin reaction. We studied the effects of substrate prepared from normal human plasma fraction Cohn IV-4, or from nephrectomized (2NX) sheep plasma, on PRC of normal and 2NX human plasmas before and after prorenin activation by acid, cold, and trypsin, and compared the results with plasma renin activities (PRA, no added substrate). Plasmas from 2NX men exhibited negligible basal PRA, indicating that very little, if any, renin had been formed from the extrarenal prorenin they contained, and suggesting the lack of an endogenous prorenin activating mechanism, or "convertase," of probable renal origin. Prorenin was demonstrable by tryptic activation, more than by acid or cold, at up to about 30% of normal. Addition of Cohn IV-4 substrate to 2NX plasma unexpectedly produced (i) a basal PRC value higher than in normal plasma, (ii) total renin values after activation by acid, cold, and trypsin that were much closer to normal values than reflected by PRA methodology, without a commensurate increase (if anything a decrease) in prorenin as a percentage of total renin estimated by all activation methods, and (iii) substantial equalization of activation effects such that trypsin was no longer more effective than acid and cold (and this was also noted with normal plasma). The skewing effect of adding Cohn IV-4 substrate on the PRC of 2NX plasma was much greater than in normal plasma, even though 2NX plasma already had an above normal level of endogenous substrate and should have been influenced less. Enhancement of PRC was very pronounced even when Cohn IV-4 was added to make up only 9% of total (endogenous + exogenous) substrate in the incubation system, suggesting that it was not the added substrate but a renin-generating contaminant that inflated the PRC. Such inflation could be blocked by adding protease inhibitors, suggesting that the responsible protease(s) acted as a prorenin "convertase" that generated new renin from renal and (or) extrarenal prorenin contributed by the added substrate, as well as by the plasma being assayed. One component of convertase could be kallikrein, which was identified by chromogenic assay, the importance of which relative to total convertase activity is unknown.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modification of the fatty acid composition of cultured human fibroblasts.

The fatty acid composition of human skin fibroblasts grown in 10% dialyzed fetal calf serum can be modified considerably by adding supplemental fatty acids to the culture medium. The degree of modification was dependent on the concentration of added fatty acid over the range tested, 2.5 X 10(-5) to 1 X 10(-4) M. At the higher concentration, the extent of the modifications was as those which can be produced in nonhuman or malignant cell lines. Although the greatest changes were produced in the neutral lipid fraction, the cellular phospholipids also exhibited appreciable modifications. The phospholipids isolated from a microsomal fraction prepared from the cell homogenate exhibited similar changes in fatty acyl composition. These findings indicate that the human fibroblast can tolerate considerable variability in fatty acid composition, even in membrane phospholipids. The triglyceride content of the cells increased when they were grown in the presence of added fatty acids, but the phospholipid and cholesterol content remained unchanged. Growth was not affected by either oleic or linoleic acids, but it was reduced up to 50% when palmitic linolenic, or arachidonic acid was added in concentrations of 5 X 10(-5) M or above. Extensive modifications in phospholipid fatty acid composition also were produced in confluent monolayers of these fibroblasts. This suggest that some membrane lipid turnover occurs even when the cultures are not rapidly growing. Fatty acid modifications also were produced in the commercially available IMR-90 strain of human lung fibroblasts, suggesting that the ability to tolerate considerable differences in fatty acid composition is not a special property of the skin fibroblast line that was isolated locally.