Spontaneous and detergent-induced vesiculation of thymocyte plasma membranes

An analog of lysophosphatidylcholine (1-dodecyl-propanediol-3-phosphocholine) which does not impair membrane-bound enzymes was used for the induction of shedding of membrane vesicles from intact calf thymocytes. Without liberation of intracellular enzymes such as lactate dehydrogenase (EC 1.1.1.27) the shedded membranes contained 15--25% of the total activity of the plasma membrane enzymes alkaline phosphatase (EC 3.1.3.1), nucleotide pyrophosphatase (EC 3.1.4.1) and gamma-glutamyl transferase (EC 2.3.2.2). Membrane-free supernatants only exhibited trace activities of these enzymes. Without further purification, the specific enzyme activities in shedded membranes were of the same order of magnitude as in purified plasma membranes prepared after nitrogen cavitation of thymocytes. Small amounts of membrane vesicles which showed a different composition could be removed without detergent. These membranes exhibited a 3-fold lower specific activity of the gamma-glutamyl transferase while that of the alkaline phosphatase and nucleotide pyrophosphatase was similar as in detergent induced membrane vesicles. Distinct differences also were found in the protein pattern. The content of total cholesterol and phospholipid in vesicles shed spontaneously or after detergent treatment was nearly identical, however, significant differences were found in the fatty acid composition of the main phospholipids. The content of polyunsaturated fatty acids (linoleic and arachidonic acid) increased in the order: spontaneously shedded membranes, detergent induced vesicles, conventional purified plasma membranes. These results are discussed in terms of the heterogeneous composition of areas of the thymocyte plasma membrane.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Heavy membrane-associated caspase 3: identification, isolation, and characterization

Heavy membrane preparations from 697 lymphoblastoid cells contain a tightly bound caspase zymogen. This heavy membrane-bound procaspase can be efficiently liberated from membrane preparations using detergents. Alternatively, the procaspase can be rapidly processed and activated from membrane preparations by caspase-1 without detergents. The activated caspase-3 was purified using affinity chromatography and characterized by amino acid sequencing and inhibitor specificity analysis. The sequence indicates that this heavy membrane bound caspase is caspase-3. The kinetic properties and inhibitor binding specificity also show that this purified caspase is enzymologically indistinguishable from cytoplasmic or recombinant caspase-3. However, the N-termini of activated heavy membrane-bound and cytoplasmic caspase-3 are slightly different; peptide sequencing data indicate that the heavy membrane caspase-3 begins at Lys 14, whereas the cytoplasmic enzyme begins at Ser 10. Implications of this structural difference are discussed.     

Multiple substrates for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite.

Paraoxonase (PON-1) is a high-density lipoprotein (HDL)-bound enzyme with activity toward multiple substrates. It hydrolyzes organic phosphate and aromatic carboxylic acid esters. It also inhibits accumulation of oxidized phospholipids in plasma lipoproteins by a mechanism yet to be determined. Therefore, we subjected apolipoprotein A-I proteoliposomes containing either 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine to oxidation by a peroxynitrite generator, SIN-1, in the presence and absence of purified PON-1. PON-1 modified the proportion of oxidation products without affecting the overall extent of PC oxidation. However, in the presence of PON-1, phosphatidylcholine isoprostanes were hydrolyzed to lysophosphatidylcholine. In addition, PON-1 hydrolyzed the phosphatidylcholine core aldehydes 1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(5-oxo)valeroyl-sn-glycero-3-phosphocholine to lysophosphatidylcholine. This hydrolysis was not affected by pefabloc, a serine esterase inhibitor. There was no detectable release of linoleate, arachidonate, or their hydroperoxy or hydroxy derivatives in the presence of PON-1. We conclude that PON-1 minimizes the accumulation of phosphatidylcholine oxidation products by the hydrolysis of phosphatidylcholine isoprostanes and core aldehydes to lysophosphatidylcholine with a serine esterase-independent mechanism.     

The animal ether phospholipid platelet-activating factor stimulates acidification of the incubation medium by cultured soybean cells

Addition of the animal ether phospholipid platelet-activating factor, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, (PAF) stimulates medium acidification in cultured soybean (Glycine max L.) cells. The pH of the medium after 8–10 hours is on the average one pH unit lower than in controls. With fusicoccin an average pH difference of 1.7 units is reached. Phospholipids, glycerol, 1-oleyl-2-acetyl-sn-glycerol, 1-0-hexadecyl-sn-glycerol, and triolein at the same concentrations as PAF had no stimulatory effect on medium acidification. The detergents CHAPS and deoxycholate lead to alkalinization of the medium whereas lysophosphatidylcholine (LPC), a detergent with structural similarity to PAF, shows no effect.Abbreviations CHAPS (3-((3-cholamylopropyl) dimethylamino)-1-propanesulfonate) - DOC deoxycholic acid - FC fusicoccin - LPC lysophosphatidylcholine - OAG 1-oleyl-2-acetyl-sn-glycerol - PAF platelet-activating factor = 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine - IAA indole-3-acetic acid     

Lyso-phosphatidylcholine is implicated in thioacetamide-induced liver necrosis

Thioacetamide is a weak hepatocarcinogen. To determine whether alterations in lysophosphatidylcholine are implicated in thioacetamide-induced hepatic necrosis, rats were injected i.p. with this agent (50 mg/Kg body weight per day) or diluent for 1, 3, 8 and 30 days. Serum catalytic activities of aminotransferases were determined. Incorporation of (32P)-orthophosphate into hepatic lysophosphatidylcholine was also evaluated in animals killed 75 minutes or 13 hours after isotope administration. Results demonstrate that: A significant increase in hepatic lysolecithin concentration occurs when a maximum level of serum aminotransferases is present. An increase of (32P)-orthophosphate radioactive incorporation in lysolecithin was observed at the two assayed labelling periods, which suggest an activation of phospholipase A. The radioactivity present in lysolecithin after 13 h isotope injection showed a close correlation with serum level of aminotransferases. From these results it can be deduced that lysolecithin is implicated in TAA-induced necrosis and may be generated by increase in either phospholipase A activity and/or synthesis.     

Nonenzymatic synthesis of glycerolipids catalyzed by imidazole

Imidazole catalyzed acylations of lysolipids by acyl-CoAs in water at room temperature and at a pH close to neutrality. In the presence of oleoyl-CoA and either lysophosphatidylcholine, 1-palmitoyl-sn-glycero-3-phosphocholine (LPC); lysophosphatidylglycerol, monoacyl-sn-glycero-3-phosphoglycerol; lysophosphatidyl acid, 1-oleoyl-sn-glycero-3-phosphate; lysophosphatidylserine, monoacyl-sn-glycero-3-phosphoserin; or lysophosphatidylethanolamine, monoacyl-sn-glycero-3-phosphoethanolamine, the corresponding phospholipids were synthesized. Similarly, the use of lyso-platelet activating factor, an ether analog of LPC, yielded the formation of 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine. In the presence of LPC, an imidazole-catalyzed synthesis of phosphatidylcholine (PC) occurred when medium, long, and very long chain acyl-CoAs were added. With hydroxyacyl-CoA, a similar PC synthesis was obtained. The process described in the present paper appears to offer several potential applications of interest for the synthesis of glycerophospholipids and triglycerides with labeled and/or an unusual or fragile fatty acid, or when suitable acyltransferases have not yet been described in the literature and/or are not commercially available. The method described is very safe and simple since lipids can be synthesized in tubes containing 0.7% imidazole in water, and left for a few hours at room temperature on the bench.     

Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle

Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed.     

Detergent properties of water-soluble choline phosphatides. Selective solubilization of acyl-CoA:lysolecithin acyltransferase from thymocyte plasma membranes.

Several analogs of lysolecithin were found to solubilize human erythrocyte ghosts comparably or even better than other detergents. Derivatives with aliphatic chains of 12 to 14 carbons were most effective. The phosphorylcholine detergents apparently possess low protein-denaturing properties, since they, for the first time, allowed the solubilization of enzymatically active acyl-CoA:lysolecithin acyltransferase from thymocyte plasma membranes. The solubilized enzyme was not sedimented at 177,000 x g for 60 min and penetrated into Sepharose 6B gels. Low detergent concentration resulted in a selective extraction of the acyltransferase (about 70%) as compared to alkaline phosphatase, nucleotide pyrophosphatase, gamma-glutamyltransferase or Mg2+-ATPase (30 to 40%). The selectivity was reflected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of soluble and sedimentable membrane fractions; three bands of approximately 53, 84, and 94 x 10(3) daltons were enriched in the supernatants, whereas one band of about 68 x 10(3) daltons was concentrated in the pellet. The preferential extraction of acyltransferase may be related to particularly high affinity of lysolecithin analogs for this enzyme, which at higher concentrations was competitively inhibited by these detergents. The inhibitor constants ranged from 1400 micron for the C10 analog (ET-10-H) to 80 micron for the compound with 16 carbons (ET-16-H) per aliphatic chain.     

Biosynthesis of pulmonary surfactant: Comparison of 1-palmitoyl-sn-glycero-3-phosphocholine and palmitate as precursors of dipalmitoyl-sn-glycero-3-phosphocholine in adenoma alveolar type II cells

Urethan-induced pulmonary adenomas of mice are composed of cells that appear to be morphologically identical to alveolar type II cells and synthesize disaturated diacyl-sn-glycero-3-phosphocholine, the major component of pulmonary surfactant. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid were compared as precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma type II cells by incubating both substrates with whole adenomas. When the precursors were compared at equal concentrations (100 μm) in the presence of albumin (1 mg/ml), the rates of incorporation of 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine and [1-¹⁴C]palmitic acid into diacyl-sn-glycero-3-phosphocholine were 5.2 and 2.9 nmol/min · g tissue, respectively. The concentration of monoacyl-sn-glycero-3-phosphocholine (lysolecithin) in the blood plasma of BALB/c mice was 150 μm. In short-term labeling experiments, the label in disaturated diacyl-sn-glycero-3-phosphocholine was equally distributed between the sn-1 and sn-2 positions when 1-[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine was the precursor, whereas 75 to 80% was in the sn-2 position when [1-¹⁴C]palmitic acid was the precursor. The ratios are consistent with incorporation of 1-palmitoyl-sn-glycero-3-phosphocholine via the lysolecithin:lysolecithin transacylase reaction and incorporation of palmitate via acylation of 1-palmitoyl-sn-glycero-3-phosphocholine by acyl-CoA:lysolecithin acyltransferase. 1-[1-¹⁴C]Palmitoyl-sn-glycero-3-phospho-[³H-methyl]choline was incorporated into total cellular diacyl-sn-glycero-3-phosphocholine with an isotope ratio similar to that of the precursor; the disaturated species was more enriched in ¹⁴C. These findings indicate the cells take up intact monoacyl-sn-glycero-3-phosphocholine and incorporate it into diacyl-sn-glycero-3-phosphocholine. The ability of the cells to utilize intact lysophosphoglycerides for synthesis of cellular lipids was further demonstrated by showing that ether analogs, 1-alkyl-sn-glycero-3-phosphocholine and 1-alkyl-sn-glycero-3-phosphoethanolamine, are taken up and acylated by the cells. Activities of lysolecithin:lysolecithin transacylase and acyl-CoA:lysolecithin acyltransferase were measured in subcellular fractions of the adenoma type II cells; the specific activities of the enzymes were 2.1 nmol/min · mg soluble protein and 21 nmol/min · mg microsomal protein, respectively. The total activity of the acyltransferase in the cell fractions was about four-fold higher than the activity of the transacylase. Characteristics of the two enzymes were studied and are discussed. The findings indicate that exogenous 1-palmitoyl-sn-glycero-3-phosphocholine and palmitic acid both serve as efficient precursors of disaturated diacyl-sn-glycero-3-phosphocholine in the adenoma alveolar type II cells.     

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to <40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.In this method, we provide comparison of the purification of CFTR in dodecyl-β-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1''-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.     

Lysolecithin as regulator of de novo lecithin synthesis in rat liver microsomes.

1-Acyl-sn-glycero-3-phosphocholine (lysolecithin) was found to affect 1,2-diacyl-sn-glycerol:CDPcholine cholinephosphotransferase (CPT; EC 2.7.8.2) activity of rat liver microsomes in a concentration dependent, characteristic manner. Cholinephosphate transfer was activated at lysolecithin concentrations below 0.5 mM with a maximum stimulation occurring at 75–100 μM lysolecithin levels. At concentrations above 0.5 mM, CPT activity was inhibited by lysolecithin. It was shown that CPT inhibition by lysolecithin is competitive (Ki ≈ 0.6 mM) with respect to CDPcholine. The possible role of lysolecithin as regulator of de novo lecithin synthesis in vivo is outlined.     

Non-ionic hybrid detergents for protein delipidation

Non-ionic detergents are important tools for the investigation of interactions between membrane proteins and lipid membranes. Recent studies led to the question as to whether the ability to capture protein-lipid interactions depends on the properties of detergents or their concentration in purification buffers. To address this question, we present the synthesis of an asymmetric, hybrid detergent that combines the head groups of detergents with opposing delipidating properties. We discuss detergent properties and protein purification outcomes to reveal whether the properties of detergent micelles or the detergent concentration in purification buffers drive membrane protein delipidation. We anticipate that our findings will enable the development of rationally design detergents for future applications in membrane protein research.     

Preparation of a photoreactive analog of parathyroid hormone [Nle8, Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]bovine parathyroid hormone-(1-34)NH2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors

Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.     

Alkyl-lysophospholipid accumulates in lipid rafts and induces apoptosis via raft-dependent endocytosis and inhibition of phosphatidylcholine synthesis

The synthetic alkyl-lysophospholipid (ALP), 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, is an antitumor agent that acts on cell membranes and can induce apoptosis. We investigated how ALP is taken up by cells, how it affects de novo biosynthesis of phosphatidylcholine (PC), and how critical this is to initiate apoptosis. We compared an ALP-sensitive mouse lymphoma cell line, S49, with an ALP-resistant variant, S49(AR). ALP inhibited PC synthesis at the CTP:phosphocholine cytidylyltransferase (CT) step in S49 cells, but not in S49(AR) cells. Exogenous lysophosphatidylcholine, providing cells with an alternative way (acylation) to generate PC, rescued cells from ALP-induced apoptosis, indicating that continuous rapid PC turnover is essential for cell survival. Apoptosis induced by other stimuli that do not target PC synthesis remained unaffected by lysophosphatidylcholine. Using monensin, low temperature and albumin back-extraction, we demonstrated that ALP is internalized by endocytosis, a process defective in S49(AR) cells. This defect neither involved clathrin-coated pit- nor fluid-phase endocytosis, but depended on lipid rafts, because disruption of these microdomains with methyl-beta-cyclodextrin or filipin (sequestering cholesterol) or bacterial sphingomyelinase reduced uptake of ALP. Furthermore, ALP was found accumulated in isolated rafts and disruption of rafts also prevented the inhibition of PC synthesis and apoptosis induction in S49 cells. In summary, ALP is internalized by raft-dependent endocytosis to inhibit PC synthesis, which triggers apoptosis.     

Solubilization of the plastidial lysophosphatidylcholine acyltransferase from Allium porrum leaves: towards plants devoid of eukaryotic plastid lipids?

To analyse the involvement of the plastidial lysophosphatidylcholine (lyso-PC) acyltransferase in the import of the extraplastidial lipid precursors required for eukaryotic plastid lipid synthesis, we plan to obtain transgenic plants. Since no sequence of lyso-PC acyltransferase is known, the purification of this enzyme has been undertaken to establish its sequence. First we determined the conditions allowing the solubilization of this membrane-bound enzyme. It is shown that by using CHAPS as a detergent, a lyso-PC acyltransferase activity is associated with the solubilized proteins.     

Synthesis and biochemical studies of analogs of platelet-activating factor bearing a methyl group at C2 of the glycerol backbone

Two platelet-activating factor (PAF) analogs containing a methyl group at C2 of the glycerol moiety were synthesized, and some of their biochemical properties were investigated. 1-O-Hexadecyl-2-C,O-dimethyl-rac-glycero-3-phosphocholine (2-methyl-2-methoxy PAF) was prepared in a synthetic scheme beginning with the etherification of 2-methylpropen-1-ol. A reaction sequence involving hydroxylation, tritylation, alkylation, and detritylation afforded 1-O-hexadecyl-2-C,O-dimethyl-rac-glycerol, which was converted into the phosphocholine. A 2-lyso derivative of this PAF analog (2-methyl-lyso PAF) was synthesized from 1-O-hexadecyl-2-C-methyl-3-O-trityl-rac-glycerol. Benzylation followed by detritylation gave 1-O-hexadecyl-2-C-methyl-2-O-benzyl-rac-glycerol, which was converted into the phosphocholine compound. Hydrogenolysis afforded 1-O-hexadecyl-2-C-methyl-rac-glycero-3-phospholine (2-methyl-lyso PAF). The 2-methyl-lyso PAF analog served as a substrate for the acetyl-CoA-dependent acetyltransferase that acetylates 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine. However, 2-methyl-lyso PAF did not have a significant effect on the activities of a CoA-independent transacylase or of the acetylhydrolase that inactivates PAF, and thus does not appear to be a substrate or an inhibitor, respectively, for these enzymes. In addition, this analog exhibited only one-half of the antitumor activity of rac-1-O-alkyl-2-methoxy-rac-glycero-3-phosphocholine in human leukemic (HL-60) cells, and elicited no hypotensive response in rats and no platelet-activating activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of surface-active agents on arylsulfatase-C of human cultured fibroblasts

Arylsulfatase-C is a microsomal membrane-bound enzyme with unusual biochemical and genetic properties. Whether it is a single enzyme hydrolyzing different sterol sulfates or a complex of enzymes, with each enzyme hydrolyzing a specific substrate, has not been resolved. Its locus has been mapped to the human X chromosome but appears to escape inactivation. As a first step to clarify its biochemical properties, a systematic search was undertaken for a suitable detergent that can release this enzyme from human cultured fibroblast membranes in a form that is biologically active and electrophoretically mobile. Four non ionic (Triton X-100, Nonidet P-40, Digitonin, and saponin) and four amphoteric (lysolecithin, Zwittergent, Miranol, and Chaps) detergents were studied. At 1% concentration, they released more than 80% of the activity into a low-speed supernatant fraction, except for Saponin which had no effect. With Triton X-100 and Miranol representing the two groups of detergents, significant release occurred only when the detergent concentrations exceeded their respective critical micelle concentrations, thus indicating that arylsulfatase-C is an integral membrane protein. The apparent molecular weight of the detergent-enzyme complex, ascertained by gel filtration, was 85,000 in the presence of Triton X-100 and 335,000 in the presence of Miranol. However, only the preparation solubilized by Miranol (and Chaps, to a lesser degree) permitted migration of the enzyme in nitrocellulose acetate during electrophoresis at pH 7.0, while the enzyme extracted with all other detergents remained at the origin. Therefore, the amphoteric detergent, Miranol, appears to fulfill the requirements for further characterization of the membrane-bound arylsulfatase-C in human cultured fibroblasts.     

Stimulation of galactosyltransferase in liver microsomes by lysolecithin

Lysolecithin markedly stimulated membrane-bound UDP-galactose:glycoprotein galactosyltransferase. The parent molecule lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidic acid, lysophosphatidic acid or glycerophosphorylcholine did not activate the enzyme suggesting that both fatty acyl- and phosphorylcholine groups are required for the enzyme activation. The dose-effect of lysolecithin showed sigmoidal kinetics and the Vmax of the enzyme was increased several-fold by lysolecithin. Saturating amounts of Triton masked the effect of lysolecithin. Pre-incubation with phospholipase A also activated the enzyme. A possible role of membrane lysolecithin is indicated in regulating the enzymes of glycoprotein synthesis.