The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.     

Purification and characterization of protein kinase C epsilon from rabbit brain.

Protein kinase C epsilon was chromatographically purified from rabbit brain to electrophoretic homogeneity. We identified the enzyme as the epsilon species of novel-type protein kinase C (nPKC epsilon), originally discovered and defined by cDNA cloning [Ohno, S., et al. (1988) Cell 53, 731-741], on the basis of the following observations: (i) the enzyme reacts specifically with an antipeptidic antiserum to nPKC epsilon but not with antisera to any of the other molecular species of PKC thus far known; (ii) it exhibits enzymatic behavior essentially identical to that of a recombinant nPKC epsilon purified from transfected COS cells [Konno, Y., et al. (1989) J. Biochem. 106, 673-678] and distinct from that of conventional PKC (alpha, beta I/II, and gamma) in its dependence on magnesium concentration and cofactors such as phospholipids, calcium, and phorbol ester; and (iii) it has an apparent molecular weight of 95.7K +/- 0.4K on SDS-PAGE, significantly greater than the other conventional and novel PKCs thus far identified. Notably, calcium exhibits a complex effect, both positive and negative, on the kinase activity of epsilon depending on the kind of substrate and the coexisting phospholipid, calling for a modification of the current notion that epsilon is a kinase unresponsive to calcium. The amount of epsilon species in the brain was estimated to be comparable to that of each conventional species, indicating that epsilon stands as one of the major PKC family members in brain. Furthermore, the enzyme shows a broader substrate spectrum than conventional PKC when examined with endogenous substrates, implying that it may cover a wider or different range of physiological functions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of angiotensin-converting enzyme inhibitory activity by acid-limited proteolysis of glyceraldehyde 3-phosphate dehydrogenase

Angiotensin-converting enzyme (ACE) inhibitors were excised from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) preparations of tuna and porcine muscles by heating at 120 degrees C for 5 min in 1 M AcOH-20 mM HCl. The inhibitors were then purified by successive chromatographies. The final product from tuna was identified as Pro-Thr-His-Ile-Lys-Trp-Gly-Asp, which was the ACE inhibitor obtained from tuna muscle [Kohama et al. (1988) Biochem. Biophys. Res. Commun. 155, 332-337]. The porcine ACE inhibitor was found to be Pro-Ala-Asn-Ile-Lys-Trp-Gly-Asp, which was identical to the porcine muscle GAPDH peptide 79-86. These results strongly suggested that the ACE inhibitory octapeptides derived from GAPDH proteins by acid-limited proteolysis at Asp-Pro and Asp-Ala peptide bonds.     

Kinetics of the hydrolysis of monodispersed and micellar phosphatidylcholines catalyzed by a phospholipase C from Bacillus cereus

The phosphatidylcholine-hydrolyzing phospholipase C, so-called "phospholipase C" (PLC), was isolated from the culture of Bacillus cereus strain IAM 1208. The amino-acid composition and partial N-terminal sequence of the purified enzyme were in good agreement with those expected from the nucleotide sequence for a PLC of strain ATCC 10987 [Johansen et al. (1988) Gene 65, 293-304]. The chain-length dependence of kinetic parameters for the PLC-catalyzed hydrolysis of monodispersed short-chain phosphatidylcholines (diCNPC, N = 3-6) was studied by a pH-stat assay method at 25 degrees C, pH 8.0, and ionic strength 0.2 in the presence of saturating amounts of Zn2+ (0.1 mM). The result was compared with those for snake venom phospholipases A2 [Teshima et al. (1989) J. Biochem. 106, 518-527]. It was found that the interaction of the PLC with the head group of the substrate molecule is very important for the binding. The pH dependences of kinetic parameters for the hydrolysis of monodispersed diC5PC and mixed micelles of diC16PC with Triton X-100 were also studied under the same conditions. An ionizable group, whose pK value is perturbed from 7.77 to 8.30 by substrate binding, was found to be essential to the catalysis. This group was tentatively assigned to His 14 on the basis of the results on X-ray crystallographic and chemical modification studies [Hough et al. (1989) Nature 338, 357-360 and Little (1977) Biochem. J. 167, 399-404].(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose-6-phosphate dehydrogenase from Dicentrarchus labrax liver: kinetic mechanism and kinetics of NADPH inhibition

The kinetic mechanism of the reaction catalyzed by glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from Dicentrarchus labrax liver was examined using initial velocity studies, NADPH and glucosamine 6-phosphate inhibition and alternate coenzyme experiments. The results are consistent with a steady-state ordered sequential mechanism in which NADP+ binds first to the enzyme and NADPH is released last. Replots of NADPH inhibition show an uncommon parabolic pattern for this enzyme that has not been previously described. A kinetic model is proposed in agreement with our kinetic results and with previously published structural studies (Bautista et al. (1988) Biochem. Soc. Trans. 16, 903-904). The kinetic mechanism presented provides a possible explanation for the regulation of the enzyme by the [NADPH]/[NADP+] ratio.     

Primary structure of a ribonuclease from porcine liver, a new member of the ribonuclease superfamily

In most tissues, ribonucleases (RNases) are found in a latent form complexed with ribonuclease inhibitor (RI). To examine whether these so-called cytoplasmic RNases belong to the same superfamily as pancreatic RNases, we have purified from porcine liver two such RNases (PL1 and PL3) and examined their primary structures. It was found that RNase PL1 belonged to the same family as human RNase Us [Beintema et al. (1988) Biochemistry 27, 4530-4538] and bovine RNase K2 [Irie et al. (1988) J. Biochem. (Tokyo) 104, 289-296]. RNase PL3 was found to be a hitherto structurally uncharacterized type of RNase. Its polypeptide chain of 119 amino acid residues was N-terminally blocked with pyroglutamic acid, and its sequence differed at 63 positions with that of the pancreatic enzyme. All residues important for catalysis and substrate binding have been conserved. Comparison of the primary structure of RNase PL3 with that of its bovine counterpart (RNase BL4; M. Irie, personal communication) revealed an unusual conservation for this class of enzymes; the 2 enzymes were identical at 112 positions. Moreover, comparison of the amino acid compositions of these RNases with that of a human colon carcinoma-derived RNase, RNase HT-29 [Shapiro et al. (1986) Biochemistry 25, 7255-7264], suggested that these three proteins are orthologous gene products. The structural characteristics of RNases PL1 and PL3 were typical of secreted RNases, and this observation questions the proposed cytoplasmic origin of these RI-associated enzymes.     

Isolation and structural characterization of low-molecular-mass monosialyl oligosaccharides derived from respiratory-mucus glycoproteins of a patient suffering from bronchiectasis

The carbohydrate chains of the respiratory-mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive straight-phase high-performance liquid chromatography (HPLC) on a silica-based alkylamine column, and reverse-phase HPLC on a silica-based octadecyl column, respectively. The structures of the oligosaccharidealditols were determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are: (formula; see text) 23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631-642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643-654]. The sialic acid residue was found to occur either in alpha (2----3)- or alpha (2----6)-linkage to a galactosyl residue or in alpha (2----6)-linkage to GalNAc-ol.     

In vivo and in vitro induction of 'tissue' transglutaminase in rat hepatocytes by retinoic acid.

Tissue transglutaminase (tTG) expression was found to be induced in rat liver following in vivo retinoic acid (RA) treatment (Piacentini et al. (1988) Biochem. J. 253, 33-38). Here we show that the increased enzyme expression in rat liver is at least partially the result of the action of RA in parenchymal cells. In fact, (a) when hepatocytes are isolated from RA-treated animals their transglutaminase protein content is much higher than in similarly isolated control cells; (b) higher tTG protein level is also found by immunoelectronmicroscopy in the hepatocytes of the RA-treated rats as compared with the very low amount detected in the controls; (c) RA induces tTG in hepatocytes under culture conditions as well. One of the functions of tTG is to form a protein polymer in dying apoptotic cells by epsilon(gamma-glutamyl)lysine and, specifically gamma-glutamylpolyamine cross-links (Fesus et al. (1989) FEBS Lett. 245, 150-154). Noteworthy, after in vivo and in vitro RA-treatment we could not determine any increase (there was even a slight decrease) in the number of the cross-linked apoptotic envelopes. In keeping with this is the significant reduction of protein bound gamma-glutamylpolyamine detected in hepatocytes exposed to RA in culture. These findings suggest that the RA-induced tTG in parenchimal cells is an inactive form.     

Identification of functionally important cysteine residues of the human renin-binding protein as the enzyme N-acetyl-D-glucosamine 2-epimerase

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353] and its active site residue was determined to be cysteine 380 by site-directed mutagenesis [Takahashi, S. et al. (1999) J. Biochem. 126, 639-642]. To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells. The expression was detected by Western blotting using anti-rhRnBP antiserum. The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity. Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity.     

Properties of catalase purified from a methanol-grown yeast, Kloeckera sp. 2201

Catalase, a marker enzyme of peroxisomes, was purified to homogeneity from whole cells of Kloeckera sp. 2201 (a strain of Candida boidinii) grown on methanol by means of ammonium sulfate fractionation followed by hydroxyapatite, Sephacryl S-300 and DEAE-Sepharose column chromatographies. Crystallized catalase was brown-coloured and needle-like. The molecular mass of the enzyme was about 240 000 daltons consisting of four identical subunits of 62 000 daltons. The minimum size of catalase molecule was estimated to be about 6 X 10 nm from an electron micrograph. Judging from the absorption spectrum, the enzyme seemed to belong to a group of T-type catalase. The Km value of the enzyme for hydrogen peroxide (catalatic activity) was 25 mM, while that for methanol (peroxidatic activity) was 83 mM. Catalase from Kloeckera sp. cells showed a certain degree of similarity to the enzyme purified from alkane-grown Candida tropicalis [T. Yamada et al. (1982) Eur. J. Biochem. 125, 517-521 and 129, 251-255] in its immunochemical properties.     

Transduction of the chemotactic signal to the actin cytoskeleton of Dictyostelium discoideum

Dictyostelium discoideum amebae chemotax toward folate during vegetative growth and toward extracellular cAMP during the aggregation phase that follows starvation. Stimulation of starving amebae with extracellular cAMP leads to both actin polymerization and pseudopod extension (Hall et al., 1988, J. Cell. Biochem. 37, 285-299). We have identified an actin nucleation activity (NA) from starving amebae that is regulated by cAMP receptors and controls actin polymerization (Hall et al., 1989, J. Cell Biol., in press). We show here that NA from vegetative cells is also regulated by chemotactic receptors for folate. Our studies indicate that NA is an essential effector in control of the actin cytoskeleton by chemotactic receptors. Guided by a recently proposed model for signal transduction from the cAMP receptor (Snaar-Jagalska et al., 1988, Dev. Genet. 9, 215-225), we investigated which of three signaling pathways activates the NA effector. Treatment of whole cells with a commercial pertussis toxin preparation (PT) inhibited cAMP-stimulated NA. However, endotoxin contamination of the PT appears to account for this effect. The synag7 mutation and caffeine treatment do not inhibit activation of NA by cAMP. Thus, neither activation of adenylate cyclase nor a G protein sensitive to PT treatment of whole cells is necessary for the NA response. Actin nucleation activity stimulated with folate is normal in vegetative fgdA cells. However, cAMP suppresses rather than activates NA in starving fgdA cells. This indicates that the components of the actin nucleation effector are present and that a pathway regulating the inhibitor(s) of nucleation remains functional in starving fgdA cells. The locus of the fgdA defect, a G protein implicated in phospholipase C activation, is directly or indirectly responsible for transduction of the stimulatory chemotactic signal from cAMP receptors to the nucleation effector in Dictyostelium.     

Genomic tagging of endogenous type IIbeta phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation

Previous studies from acutely transfected HeLa cells have identified an acidic alpha-helix in the Type IIbeta PtdIns5P 4-kinase (PIPkin IIbeta) as a putative novel nuclear localisation sequence (Ciruela et al. Biochem. J. 364, 587-591 2000). However, some heterogeneity in cellular localisation was always observed, and other published aspects of PIPkin IIbeta physiology are more consistent with an extra-nuclear function. As a means of resolving whether the endogenous PIPkin IIbeta is nuclear, we have used the high homologous recombination frequency of DT40 cells to knock an epitope tag (Mosedale et al., Nat Struct Biol. 12, 763-771 2005) into one of the alleles of the DT40 PIPkin IIbeta gene. We show that PIPkin IIbeta is expressed as a tagged protein, is active as revealed by immunoprecipitation and enzyme assay, and that cellular fractionation reveals that it is indeed nuclear. Genomic tagging of endogenous proteins in DT40 cells is a technique that offers unique advantages in studying endogenous signalling proteins.     

Localization of an endogenous ADP-ribose acceptor, p33, in polymorphonuclear cell granules in chicken liver interlobular connective tissue

We investigated immunohistochemically the localization of p33, an endogenous substrate protein for an arginine-specific ADP-ribosyltransferase in chicken liver. Polymorphonuclear-pseudo-eosinophilic granulocytes (heterophils) in interlobular connective tissues of the liver were exclusively and strongly stained with the antibody against p33. Strong reactivity was associated with granules in cytoplasm of the heterophils. When the chicken liver nuclear fraction was washed, the transferase activity was released into the 600 x g supernatant fraction while a nuclear enzyme poly(ADP-ribose) synthetase was retained in the pellet fraction. These results indicate that p33 and probably also ADP-ribosyltransferase, found in the liver nuclear fraction [Tanigawa et al. (1984) J. Biol. Chem. 259, 2022-2029, Mishima et al. (1988) Eur. J. Biochem. 179, 267-273], originate from interlobular heterophils of the chicken liver.     

The recombinant spinach acyl-acyl carrier protein-I expressed in Escherichia coli is the 18:1 delta 11(cis) thioester

A synthetic spinach acyl carrier protein-I (ACP-I) gene was cloned and expressed in the Escherichia coli beta-alanine auxotroph SJ16 (P. D. Beremand et al. (1987) Arch. Biochem. Biophys. 256, 90-100). After characterization of the transformed cells and purification of the protein product it was evident that 50% of the recombinant spinach ACP-I was acylated during early log-phase growth (D. J. Guerra et al. (1988) J. Biol. Chem. 263, 4386-4391). We have purified the recombinant acyl-acyl carrier protein-I to greater than 90% homogeneity and have made a fatty acid methyl ester of the delipidated and trypsin-treated preparation. We have found that the acyl moiety attached to recombinant spinach acyl carrier protein-I is 18:1 delta 11(cis) (cis-vaccenic acid) a major unsaturated end product of Escherichia coli de novo fatty acid synthesis. This result reflects previous work (D. S. Guerra et al. (1986) Plant Physiol. 82, 448-453) which suggested the acyl carrier protein-I structure has evolved from ancestral ACP structures to accommodate the eukaryotic pathway of lipid synthesis in higher plants. The accumulation of recombinant 18:1 delta 11(cis) acyl carrier protein-I in transformed E. coli SJ16 cells attests to the poor reactivity of this substrate to acyl transferase reactions and may help explain the lack of effect on pools of fatty acids found in vivo.     

Biosynthesis of dehydro-N-acetyldopamine by a soluble enzyme preparation from the larval cuticle of Sarcophaga bullata involves intermediary formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide

The enzymes involved in the side chain hydroxylation and side chain desaturation of the sclerotizing precursor N-acetyldopamine (NADA) were obtained in the soluble form from the larval cuticle of Sarcophaga bullata and the mechanism of the reaction was investigated. Phenylthiourea, a well-known inhibitor of phenoloxidases, drastically inhibited both the reactions, indicating the requirement of a phenoloxidase component. N-acetylcysteine, a powerful quinone trap, trapped the transiently formed NADA quinone and prevented the production of both N-acetylnorepinephrine and dehydro NADA. Exogenously added NADA quinone was readily converted by these enzyme preparations to N-acetylnorepinephrine and dehydro NADA. 4-Alkyl-o-quinone:2-hydroxy-p-quinone methide isomerase obtained from the cuticular preparations converted chemically synthesized NADA quinone to its quinone methide. The quinone methide formed reacted rapidly and nonenzymatically with water to form N-acetylnorepinephrine as the stable product. Similarly 4-(2-hydroxyethyl)-o-benzoquinone was converted to 3,4-dihydroxyphenyl glycol. When the NADA quinone-quinone isomerase reaction was performed in buffer containing 10% methanol, beta-methoxy NADA was obtained as an additional product. Furthermore, the quinones of N-acetylnorepinephrine and 3,4-dihydroxyphenyl glycol were converted to N-acetylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively, by the enzyme. Comparison of nonenzymatic versus enzymatic transformation of NADA to N-acetylnorepinephrine revealed that the enzymatic reaction is at least 100 times faster than the nonenzymatic rate. Resolution of the NADA desaturase system on Benzamidine Sepharose and Sephacryl S-200 columns yielded the above-mentioned quinone isomerase and NADA quinone methide:dehydro NADA isomerase. The latter, on reconstitution with mushroom tyrosinase and hemolymph quinone isomerase, catalyzed the biosynthesis of dehydro NADA from NADA with the intermediary formation of NADA quinone and NADA quinone methide. The results are interpreted in terms of the quinone methide model elaborated by our group [Sugumaran: Adv. Insect Physiol. 21:179-231, 1988; Sugumaran et al.: Arch. Insect Biochem. Physiol. 11:109, 1989] and it is concluded that the two enzyme beta-sclerotization model [Andersen: Insect Biochem. 19:59-67, 375-382, 1989] is inadequate to account for various observations made on insect cuticle.     

Enzymatic activities of P-450(11 beta)s expressed by two cDNAs in COS-7 cells

Expression plasmids were constructed using two cDNA clones of P-450(11 beta), pcP-450-(11 beta)-2, and pcP-450(11 beta)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11 beta) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11 beta)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11 beta)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11 beta)-3 was greater than that by P-450(11 beta)-2.     

Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes

One of the two component proteases of the hatching enzyme of the fish, Oryzias latipes, low choriolytic enzyme (LCE), was isolated from the hatching liquid and partly characterized. The enzyme was a basic protein with molecular weight of about 25.5 kDa. Like high choriolytic enzyme (HCE), the other component of the O. latipes hatching enzyme [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], LCE was considered to be a zinc-protease from the results of inhibitor studies and metal analyses. However, LCE was found to be distinct from HCE not only in some biochemical characteristics such as molecular weight, amino acid composition, and isoelectric point, but also in some enzymological properties such as substrate specificity, heat stability, and mode of action toward their natural substrate, chorion (egg envelope). Although LCE was almost incapable of digesting the inner layer of intact chorion, it very efficiently digested the inner layer of chorion that had been swollen previously by the action of HCE. Taking account of the fact that HCE swells the inner layer of intact chorion by partial proteolysis but does not efficiently digest the swollen chorion any more [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], the present results demonstrated an essential role of LCE in choriolysis, in cooperation with HCE.     

Chicken muscle aldose reductase: purification, properties and relationship to other chicken aldo/keto reductases

An enzyme that catalyzes the NADPH-dependent reduction of a wide range of aromatic and hydroxy-aliphatic aldehydes was purified from chicken breast muscle. This enzyme shares many properties with mammalian aldose reductases including molecular weight, relative substrate specificity, Michaelis constants, an inhibitor specificity. Therefore, it seems appropriate to call this enzyme an aldose reductase (EC 1.1.1.21). Chicken muscle aldose reductase appears to be kinetically identical to an aldose reductase that has been purified from chicken kidney (Hara et al., Eur. J. Biochem. 133, 207-214) and to hen muscle L-glycol dehydrogenase (Bernado et al., Biochim. biophys. Acta 659, 189-198). The association of this aldose reductase with muscular dystrophy in the chick is discussed.     

Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.     

Amino-terminal amino acid sequence and chemical and functional properties of a membrane attack complex-inhibitory factor from human erythrocyte membranes

The protein corresponding to P-18 (Sugita et al. (1988) J. Biochem, 104, 633-637) was isolated from native human erythrocyte, and newly designated membrane attack complex-inhibitory factor (MACIF). The amino-terminal sequence of this protein was determined to be Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr. Endoglycosidase F digestion of MACIF decreased its molecular weight by about 6K on SDS-PAGE. On the other hand, endoglycosidase H, neuraminidase, or endo-alpha-N-acetylgalactosaminidase treatment had no effect on the molecular weight, indicating that MACIF has complex-type N-linked oligosaccharide chains, but no O-linked chain. MACIF was highly resistant against trypsin digestion and heat treatment. The inhibitory activity of MACIF on the hemolysis of EC5-8 cells was comparable to that on EC5-7 cells, indicating that MACIF inhibited the binding of C9 to the intermediate cells, or the subsequent C9 polymerization.