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An enzyme (amine:acetyl coenzyme A acetyltransferase, EC 2.3.1.5) was partially purified from the 250 000 × g supernatant of extracts of the yeast Hansenula ciferri. It catalyzed the transfer of the acetyl group of acetyl-CoA to long-chain primary amines of 6–16 carbon atoms, as well as to water-soluble primary amines, such as glucosamine, histamine, tryptamine, serotonine, hydroxytyramine and noradrenaline, but not to polyamines, amino acids or sphingosine bases. The acetylation of the long-chain amines was inhibited by detergents, coenzyme A, butyryl or palmitoyl coenzyme A and by SH-inhibitors. Hyperbolic curves were obtained when reaction rates were measured as a function of increasing acetyl CoA concentrations, while maintaining a constant concentration of the amine. The curves were also hyperbolic when acetyl-CoA was maintained at a constant level and the concentrations of the water-soluble amines or normal amines of 6–10 carbon atoms were varied. With long-chain amines of 12–16 carbon atoms as the varying substrates, the v/S curves were hyperbolic to a certain concentration of the amine, above this concentration (“inversion point”) reaction rates decreased. The inversion points of the three amines were close to their “critical micellar concentration”. Serum albumin increased the reaction rates as well as the Km values with the long-chain amines but not with the water-soluble compounds. These data suggest that the enzyme utilizes molecular solutions of the amines but is inhibited by their micellar aggregates.

The enzymatic lesions responsible for the accumulation of acetylated sphingosine bases in the “high producer” strains of the genus Hansenula were studied. Two strains, one a “low” and the other a “high” producer, were selected to compare four enzymatic steps leading to the synthesis of acetylated dihydrosphingosine by yeast microsomes. Both strains were very similar in their generation time and DNA content, but the high producer accumulated 150 times more sphingosine bases than the low producer. Among the four microsomal enzymes, the specific activity of the palmityl thiokinase and 3-ketodihydrosphingosine reductase were almost identical in the two strains. In contrast, the specific activity of the 3-ketodihydrosphingosine synthetase, which catalyzed the rate-limiting step in dihydrosphingosine biosynthesis, was 5–10-fold greater in the high producer strain than in the low producer. The long-chain base–acetyl-CoA acetyltransferase was at least 30 times more active in the high as compared with the low producer. It therefore seems that the latter two enzymes may be the cause for the in vivo accumulation of acetylated sphingosine bases in the high producers.

1. 1. The contents of free and acetylated sphingosine bases of 25 yeast strains of the genus Hansenula were determined. On the basis of their sphingosine base content, these strains could be divided into three main subgroups: a, low producers which had 2–5 μmoles sphingosine bases per 1 of growth medium; b, intermediate producers (10–40 μmoles per 1) and high producers (120–260 μmoles per 1). 2. 2. All intermediate and high producers belonged to the species Hansenula ciferri. Several strains of this species as well as all species other than H. ciferri were low producers. 3. 3. The intermediate and high producers incorporated radioactivity of 3H or 14C-labelled serine, glucose, palmitate or acetate into sphingosine bases at rates 15–500 × higher than the low producers. 4. 4. There was no correlation between sphingosine base content and the following : Growth rate, DNA content, oxygen uptake, CO2evolution, incorporation of [3H]-leucine and [3H]serine into cell proteins, incorporation of glucose into total fatty acids, as well as levels of palmitic acid thiokinase, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase. 5. 5. An enzyme which acetylates sphingosine bases (sphingosine-acetyl CoA acetyl transferase) was present in the microsomes of the high and intermediate producers but was practically absent from those of the low producers. 6. 6. The results suggest that accumulation of sphingosine bases in the intermediate or high producers is due to increased rates of biosynthesis; the free bases are subsequently acetylated by acetyl-CoA, a reaction catalyzed by the enzyme sphingosine-acetyl-CoA acetyl transferase.

Microsomes of the yeast, Hansenula ciferri NRRL Y-1031, F-60-10, contain an enzyme (long chain base-acetyl coenzyme A acetyltransferase, EC 2.3.1) which catalyzes the transfer of the acetyl moiety of acetyl coenzyme A to both the amino and hydroxyl groups of the sphingosine bases; a separation of these two transfer reactions could not be achieved. The enzyme also catalyzed an acetyl transfer to the hydroxyl groups of N-acetylated sphingosine bases and to the amino groups of normal aliphatic primary amines of 6 to 18 carbon atoms, but not to compounds such as p-nitroaniline, glucosamine, secondary amines, or primary alcohols. A similar enzyme was not found in rat organs nor in extracts of pigeon liver acetone powder. The reaction rates were directly proportional to enzyme concentration and time of incubation. The optimal pH depended on the type of buffer used. The reaction rates could be correlated to the charge conferred upon the micelles of the bases by the various buffers. The reaction was not reversible and an exchange between the products and substrates could not be shown. It was inhibited by coenzyme A but not by the lipid products.

Autocorrelation spectroscopy of Rayleigh scattered light has been used to measure the diffusion coefficients of mixed micelles of Triton X-100 with sphingomyelin. Sphingomyelin is a water-insoluble lipid. It forms a solution of mixed micelles only for molar fractions of Triton X-100 above 0.32. Over the range of Triton X-100 molar fractions from 0.32–1.0 the diffusion coefficient increases from 2.6·10−7–4.5·10−7 cm2/s, indicating that the increased concentration ratio of the detergent systematically reduces the size of the mixed micelles. Diffusion coefficients of sonicated liposomes of sphingomyelin were also measured. For specimens of unhydrogenated and partially hydrogenated sphingomyelin, respective diffusion coefficients of 8.8·10−8 and 3.1·10−8 cm2/s were obtained. The corresponding Stokes radii for the two specimens are 250 and 700 A, respectively.

Publisher Summary This chapter discusses the determination of amine acetyltransferase from the soluble fraction of Hansenula ciferri . This enzyme transfers the acetyl group of acetyl coenzyme A to an amine acceptor. The acetyltransferase from the soluble fraction of the yeast H. ciferri utilizes water-soluble amines but also acetylates normal amines up to hexadecylamine. Three assay methods are used. In two (one for the water-soluble and the second for the long-chain amines) radioactively labeled acetyl-CoA is used and the radioactivity of the acetylated amino is determined. In the third, the reaction is terminated, DTNB [5,5-dithiobis(2-nitrobenzoic acid) ] is added and the color resulting from the coenzyme A released is read. For the purification procedure, Hansenula ciferii can be obtained from the American type collection. The lyophilized culture is transferred to 100 ml erlenmeyers containing 40 ml of a yeast maintenance medium consisting of 0.3% malt extract, 0.3% yeast extract, 0.5% peptone, and 1% glucose. The purified enzyme is directly proportional to enzyme concentration up to at least 12 μg of protein and incubation time of 20–30 min. The linearity with time is improved by increasing the concentration of glycerol. Using the high-speed supernatant, the curves describing the rate as a function of enzyme concentration are not linear, and the specific activities are not constant, but increase on dilution of the enzyme with PG.

Physical properties of the hydrocarbon region in lipid bilayers were studied in a series of liposomes of lecithin and sphingomyelin containing different concentrations of dicetylphosphate. The technique used was described previously and is based on fluorescence polarization analysis of a specific probe embedded in the analyzed region. The two probes employed in this study were perylene and 1,6-diphenyl-1,3,5-hexatriene, which simulate a rotating disc and a rotating rod, respectively. The determined dynamic properties of the hydrocarbon region in the lecithin liposomes differ markedly from those of the sphingomyelin liposomes. The hydrocarbon region of the lecithin liposomes is of an invariant phase between 0° and 60° characterized by a microviscosity at 25°, η (25°), of 0.8 ± 0.1 poise and a fusion activation energy, ΔE, of 8 ± 2 Cal per mole. In contradistinction to lecithin, the hydrocarbon region of the sphingomyelin liposomes displays a distinct phase transition at 32° ± 2°. The phase at temperatures above the transition point, presumably a liquid crystalline phase, is characterized by ΔE = 16 ± 4 Cal per mole, whereas the phase below it, presumably a gel state, possesses a ΔE value lower than 3 Cal per mole. In addition to that, the·hydrocarbon layer in sphingomyelin liposomes is much more viscous than in lecithin liposomes as shown by η (25°) = 6 ± 1 poise. All of the above characteristics are only slightly and irregularly affected by the presence of dicetylphosphate, despite the strong effects it exerts on the surface charge potential of the liposomes. This indicates that the forces which dictate the dynamic properties of the hydrocarbon region in lipid bilayers predominantly originate from hydrophobic interactions.

A simple and convenient synthesis of cholesterol esters from cholesterol and fatty acid anhydrides is described. The high, reproducible yield of cholesterol ester and the small excess of fatty acid used makes this method attractive for the preparation of cholesterol esters using difficulty obtained fatty acids.

Circular dichroism studies on bovine brain sphingomyelin show the presence of a strong negative cotton effect below 200 nm, the position and magnitude of which depend on the physical state of the lipid. This cotton effect is thought to arise from the π-π ∗ transition of the amide group in the sphingomyelin backbone. The sphingomyelin contribution to the observed ellipticity of membranes and lipoprotein complexes depends on the mol fraction of amide groups present as sphingomyelin: this contribution is calculated to be less than 2% in the case of serum high density lipoprotein and the order of 20% below 200 nm in the case of the erythrocyte ghost membrane. Due to the similarity of the CD spectrum of sphingomyelin to that of a random coil polypeptide, use of uncorrected ellipticity data is expected to lead to an overestimate of the random coil content of proteins in systems containing a high sphingomyelin content.

Publisher Summary This chapter discusses the determination of long-chain base acetyltransferase from the microsomes of Hansenula ciferri . This enzyme transfers the acetyl portion of acetyl coenzyme A to the amino groups of primary amines of 6–18 carbon atoms and to the amino and hydroxyl groups of sphingosine base. The enzyme differs from the arylamine acetyltransferase of the soluble fraction of H. ciferri or of mammalian and avian liver. The transfer reaction is directly proportional to enzyme concentration (up to at least 1 mg of microsomal protein) and to the reaction time (for at least 2 hours). The optimal pH of the reaction depends on the type of buffer used; this is related to the charge imparted to the micelles of the base. The curves that describe the rate of acetyl transfer as a function of the concentration of acetyl-CoA (while maintaining the base at a fixed concentration) are hyperbolic. Similar curves, in which aeetyl-CoA is the fixed and dihydrosphingosine or hexadecylamine are the variable substrates, have a nonsymmetric sigmoidal shape. Serum albumin increases the reaction rates, and at an optimal molar ratio of albumin to base of about 0.5 the V/S curves are hyperbolic. Two assay methods are used for the determination of long-chain base acetyltransferase. In one, radioactively labeled acetyl-CoA is used and the radioactivity of the acetylated base is determined. In the second, the reaction is run in the presence of DTNB [5,5-dithiobis-(2-nitrobenzoic acid)] and the color resulting from the coenzyme A released is read.

The sedimentation coefficients of mixed micelles of Triton X-100 and sphingomyelin have been measured for Triton molar fractions between 0.32 and 0.79. These measurements, together with previously reported diffusion coefficients, are used to calculate the molecular weights of the mixed micelles, and their aggregation numbers. It is found that the molecular weight decreases steadily with increasing Triton molar fraction. However, the Triton aggregation number remains nearly constant at about 196, so that the decreasing molecular weight is reflected in the sphingomyelin aggregation number, which decreases from 442 to 50 as the Triton molar fraction increases from 0.32–0.79. Intrinsic viscosities of the mixed micelle solutions are found to be independent of Triton molar fractions, with a value of 5.5 ml/g. From these measurements a number of inferences are drawn regarding the micellar shape and structure.

Publisher Summary The three main sphingosine bases of animal tissues are sphingosine (CH3-(CH2)12-CH=CH-CHOH-CHNH2-CH2OH) and dihydrosphingosine (CH3-(CH2)14-CHOH-CHNH2-CH2OH), while in plants phytosphingosine (CH3-(CH2)13-CHOH-CHOH-CHNH2-CH2OH) predominates. Similar bases with smaller or greater chain length have been described. These bases are released from the phospho- or glycosphingolipids by acid or alkaline hydrolysis. Such hydrolyses yield mixtures of the bases as well as several derivatives which are formed as a result of the chemical reaction. The reaction medium must therefore be subjected to a purification step in which the individual bases are separated from the above mixture. In this procedure silica gel chromatography is used to separate, on a preparative scale, the pure bases from acid hydrolysis reaction mixtures. This is here exemplified by two experiments. In the first, pure sphingosine and dihydrosphingosine were obtained from the hydrolyzate of bovine spinal cord sphingolipids. In the second, phytosphingosine and dihydrosphingosine were isolated from the hydrolysate of the acetylated bases secreted into the growth medium of the yeast Hansenula ciferri.

A cell-free preparation of the yeast, Hansenula ciferri catalyzed the transfer of the acetyl group of acetyl coenzyme-A to the sphinosine bases at both their amino and hydroxyl groups. The enzyme also transferred acetyl groups to the hydroxyls of the N-acetylated sphingosine bases as well as to the amino groups of primary amines of ten or more carbon atoms. A mixture of acetate, ATP and coenzyme-A could be employed instead of acetyl CoA. The reaction had an optimal pH at about 7.8 and was inhibited by free coenzyme-A.

The recent five to eight years have been characterized by major advances in the enzymology of the complex lipids. This might be exemplified by the characterization of the enzymes which synthesize and degrade the sphingolipids; the identification of separate phospholipases Al and A2; the identification of a new pathway for phosphoglyceride biosynthesis via acyl dihydroxyacetone phosphate and further recognition of the enzymes which synthesize and degrade the glycerophosphatides. A further major advance, pertinent to this symposium, is the recognition that the sphingolipid storage diseases are caused by defects in degradative enzymes. In contrast to this rapid development, identification of the mechanisms of action of these enzymes has been extremely slow. Only scanty information is available to date on the mode of interaction of the enzymes with their respective lipid substrates, either in vivo or in vitro.

The mechanisms of the intrapancreatic formation of a myocardial depressant factor (MDF) were investigated in incubated pancreatic homogenates and in subcellular fractions. An incubation period of 90–120 min was found to be optimal for MDF production. The optimal pH for MDF production in pancreatic homogenates was found to be 5–6. MDF production required the presence of salts, but was not influenced by an anaerobic environment. Large concentrations of trypsin (i.e., 1–2 mg/ml) but not of chymotrypsin, lipase, or phospholipase A, inhibited MDF formation. Differential centrifugation of homogenates yielded several fractions which formed MDF upon incubation. Measurement of marker enzymes revealed that fractions containing lysosomes and zymogen granules produced MDF whereas fractions containing predominantly either mitochondria or microsomes did not. Moreover, addition of a sulfhydryl blocking agent (e.g., PCMB) to incubated subcellular fractions and homogenates prevented MDF formation. These data implicate the proteases of lysosomes and zymogen granules as enzymes which participate in the formation of MDF, although the precise mechanism is not completely known.

A method for measuring glucose release from liposomes by means of an oxygen electrode is described. The glucose concentration is determined from the initial rates of oxygen reduction catalysed by glucose oxidase. The reduction is directly proportional to either glucose (0.066–3.0 mM) or enzyme concentration (0.3–10 μg). The enzyme does not penetrate lipid bilayers and free glucose can be measured directly. Trapped glucose can be released and measured in the presence of detergents which do not interfere with the assay. By using appropriate calibration curves, glucose concentrations could be determined under extreme conditions of pH (3–9.5), temperature (10–51 °C), and in the presence of various salts and buffers. The application of polarographic methods for studies of the permeability of amino acids and other sugars is discussed.