Multiple forms of acetylcholinesterase from human erythrocytes

1. Acetylcholinesterase from human erythrocytes was solubilized with Triton X-100 in strong salt solution and partially purified by (NH(4))(2)SO(4) fractionation. This preparation showed three main bands of enzyme activity after electrophoresis on polyacrylamide gel and incubation with either alpha-naphthyl acetate or acetylthiocholine as enzyme substrate. Two of the multiple forms were completely inhibited by 10mum-eserine and one only partially. Treatment with neuraminidase had no effect on the electrophoretic pattern; therefore sialic acid does not appear to determine or affect the ratios of the acetylcholinesterase multiple forms, unlike those of the serum cholinesterase. 2. Chromatography of the preparation on Sephadex G-200 revealed one major peak of enzyme activity and a suggestion of two minor zones of mol.wt. 546000, 184000 and 93000 (i.e. in the proportion 6:2:1). The main peak was almost completely separated from the Triton X-100 and the overall purification was about 600-fold. Further attempts to purify the enzyme by absorption on calcium phosphate gels were unsuccessful. 3. Electrophoresis of the enzyme preparation on a polyacrylamide gradient for 24h revealed three main bands that corresponded to the three values for molecular weights obtained by column chromatography. After 70h of electrophoresis a further three zones of activity developed making six molecular entities, the molecular weights of which were simple multiples of a monomer, thus resembling the cholinesterase found in serum.     

A protease from Legionella pneumophila with cytotoxic and dermal ulcerative activity

Abstract Culture supernatants of Legionella pneumophila , Philadelphia 1, were found to have proteolytic activity, as well as a nondialyzable, heat-labile cytotoxin for Chinese hamster ovary (CHO) cells, and a factor which caused hemorrhagic dermal ulceration when injected intradermally into mice. A protease was purified from culture supernates by filtration on Sephacryl S-200 followed by chromatography on DEAE cellulose. Proteolytic activity had a pH optimum of 5.5, and migrated as two bands in PAGE, with molecular weights of 42 and 31 kDa. CHO cell cytotoxic, dermal ulcerative, and proteolytic activities copurified. The results are consistent with the same protein being responsible for these activities.     

Purification and properties of an acid deoxyribonuclease from rat small intestinal mucosa

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.     

The thrombin-like enzyme from Bothrops atrox snake venom. Properties of the enzyme purified by affinity chromatography on p-aminobenzamidine-substituted agarose.

The trhombin-like activities from the snake venoms of two subspecies of Bothrops atrox, moojeni (type I) and marajoensis (type II), were purified to homogeneity by affinity chromatography on a support consisting of the inhibitor, p-aminobenzamidine, linked to Sepharose 4B with a spacer of diaminodipropylaminosuccinate. At room temperature the enzyme was not bound to the affinity support but rather was retarded in relation to the unbound protein. As a result the thrombin-like activity eluted in a large volume following the main protein fraction. However, at 4 degrees the enzyme was absorbed to the affinity support and could be eluted specifically with the ligand benzamidine (0.15 M). Optimal conditions for column loading and washing were 0.05 M Tris.HCl/0.4 M NaCl, pH 9.0 at 4 degrees. The type I enzyme isolated in this manner showed a single major band on pH 8.9 disc gel electrophoresis as well as two minor bands. Further purification by isoelectric focusing yielded one major and two minor components. All three protein fractions had identical thrombin-like activities and amino acid composition. The major band had a specific activity of 210 to 230 NIH thrombin units/mg, a S20, w of 2.65 S, a molecular weight of 29,000, and an E1% 280 of 15.6. This protein has a carbohydrate content, measured as hexose, glucosamine, and sialic acid, of 27%. From the amino acid and carbohydrate composition a partial specific volume of 0.700 ml/g was calculated. The type I enzyme, purified on affinity chromatography only, did not activate Factor XIII and was free of thromboplastin-like activity. The type II enzyme behaved very differently from the type I on pH 8.9 polyacrylamide disc gels yielding two major bands and two minor bands. The relative amounts of these four bands were not a function of purity. The type II enzyme had a specific activity of 650 to 700 NIH thrombin units/mg, a S20, w of 2.60, and a molecular weight of 31,400.     

Further studies of the structure of human placental acid alpha-glucosidase

Acid alpha-glucosidase has been purified from human placenta to a specific activity of approximately 6800, (4-methylumbelliferyl-alpha-D-glucoside as a substrate) or 55,400 mumol g-1 min-1 (glycogen or maltose as substrate). The purified enzyme gives rise to multiple protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), i.e., a major doublet of 82K and 69K , a minor doublet of 25K and 21K , and a faint band of 100K. All of the molecular weight species stained as glycoproteins with an intensity apparently proportional to their protein content, and were present in enzyme from individuals homozygous for the allozyme alpha-Glu 1. Isoelectric focusing revealed only enzymatically active proteins which, when analysed by SDS-PAGE, gave rise to multiple molecular weight species. Chromatography of I125-labeled, purified enzyme on Bio-Gel P-100 revealed only a radiolabeled, high-molecular-weight species which corresponded with enzyme activity. These findings suggest that, in the native state, the mature enzyme exists as a high-molecular-weight species, which is dissociable in SDS to several low-molecular-weight species. These results are consistent with reports that a 100K primary product of translation is post-translationally modified to yield polypeptides of lower molecular weights, and that all of the molecular species are absent in cells genetically deficient for acid alpha-glucosidase. The possibility that the low-molecular-weight (20- 25K ) protein bands in SDS-gels corresponded to a previously reported low-molecular-weight species generated by treatment with guanidine-HCl was investigated. The I125-labeled, purified acid maltase was dissociated by guanidine into two equal peaks of approximately 64K and 28K molecular weight. Surprisingly, both peaks, when analyzed on SDS-gels, yielded identical and equally intensely staining bands of 64K molecular weight. These results suggest that the mature acid alpha-glucosidase is made up of polypeptides which are bonded in the native state by at least two different types of interaction, one type which is dissociable in SDS and one type which is dissociable in guanidine but not in SDS. The nature and possible function of the 25K polypeptide generated only by guanidine-HCl remains to be determined.     

Partial purification and characterization of a factor which stimulates an increase in ornithine decarboxylase activity in the liver from tumor cell-free ascites fluid

A factor responsible for stimulating an increase in ornithine decarboxylase activity in the liver of mice was found in tumor cell-free ascites fluid of mice 3 days after inoculation of tumor cells. The factor was purified about 70-fold in 25% yield from tumor cell-free ascites fluid. As little as 1 μg of protein of purified fraction, injected intraperitoneally into normal mice, significantly increased the activity of ornithine decarboxylase in the liver. The most active preparation of the factor formed two major protein bands on analytical polyacrylamide gel electrophoresis and both these bands stained with periodic acid-Schiff's reagent. The factor was a heat-labile, alkaline-stable, acidic protein with a molecular weight of more than 300 000. It was inactivated by treatment with 10 mM dithiothreitol, 5M urea, pronase or mixed glycosidase, but was stable on treatment with DNAase, RNAase or neuraminidase.     

Selenium-containing peroxidases of germinating barley

Germinating barley grown on an artificial medium was exposed to⁷⁵Se-selenite for 8 d. Then the leaves were homogenized and proteins were separated by means of Sephadex G-150 filtration, followed by DEAE-Sepharose chromatography. Each fraction collected was assayed for total protein, radioactivity, and peroxidase activity. In barley leaves, three protein peaks (peaks no. I, II, and III) with peroxidase activity could be separated by Sephadex G 150 filtration. Each fraction was then further separated on DEAE-Sepharose chromatography. Thus, peaks I and II were resolved by DEAE-Sepharose into one major and two minor peaks of radioactivity. However, only the major peak showed peroxidase activity. Peak III was resolved from the gel filtration on the DEAE-sepharose into one major and four minor peaks of radioactivity. The major and three of the minor radioactivity peaks contained peroxidase activity. The protein fractions were separated by polyacrylamide gel electrophoresis. The molecular weights of separated proteins were estimated by means of molecular markers, and⁷⁵Se radioactivity was evaluated by autoradiography. Thus, gel filtration peak I contained four bands with mol wts of 128, 116, 100, and 89 kDa. Of these, the 89 kDa protein contained selenium. Peak II contained three protein bands, with mol wts 79.4, 59.6, and 59.9. The 59.6 band was a selenoprotein. Peak III contained four protein bands (and some very weak bands). The four major bands had mol wts of 38.6, 31.6, 30.2, and 29.2 kDa. The last mentioned band was a selenoprotein.     

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.     

The major endogenous bovine brain protein kinase C inhibitor is a heat-labile protein.

A crude cytosolic fraction prepared from bovine brain contained protein kinase C, as shown by immunoblotting, but its activity was undetectable, suggesting the presence of interfering factors. Phosphatase, ATPase and protease activities did not account for the absence of detectable protein kinase C activity. The major contributing factor was found to be a heat-labile protein which was separated from the kinase by ion-exchange chromatography. The contribution to the total inhibitory activity of heat-stable proteins was relatively minor, suggesting that they may not function physiologically as protein kinase C inhibitors.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the rabbit proacrosin-acrosin system

A single molecular form (Mr = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 117;21-11728) was initially converted by autoactivation into an acrosin (Mr = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities (Rm = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands (Rm values of 0.395-0.412 and 0.497-0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the Mr 34,000 form of acrosin. This property was not shown by Mr 68,000 acrosin. Initial acrosin (Mr = 68,000) was activated by divalent cations such as Ca2+ and Mg2+. The enzyme was inhibited by Zn2+, Fe2+, Hg2+, and sulfhydryl blockers such as 5,5'-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn2+ is involved in acrosomal stabilization. The initial rabbit acrosin (Mr = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.     

Purification and properties of nitrate reductase from Mitsuokella multiacidus

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

Purification and further characterization of enteropeptidase from porcine duodenum

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.     

Effect of tunicamycin on molecular heterogeneity of colony stimulating factor in cultured mouse mammary carcinoma FM3A cells.

Granulocyte and macrophage colony stimulating factors obtained from cultured mouse mammary carcinoma FM3A cells showed heterogeneity in molecular size giving rise to a major component with an apparent molecular weight of 80,000 and a minor one with that of 35,000 on Sephadex G-200 column chromatography. In the presence of tunicamycin, a specific inhibitor of asparagine-linked glycosylation, the colony stimulating factor was produced normally and consisted of a single component with an apparent molecular weight of 30,000.These data indicate that the sugar moiety is not essential for the production or activity of colony stimulating factor and that the heterogeneity in molecular size of the colony stimulating factor mainly resulted from tunicamycin-sensitive glycosylation.     

A platelet factor stimulating the proliferation of vascular endothelial cells: Partial purification and characterization

Platelets have been shown to contain a novel growth factor that stimulates the proliferation of vascular endothelial cells in vitro. The factor potently stimulated both DNA synthesis and proliferation rate in serum-deprived endothelial cells. Gel exclusion chromatography showed at least two peaks of activity on endothelial cells, the major peak being at an apparent molecular weight of 20 000. Isoelectric focusing revealed that the pI of the factor was 4.0-4.8. It was adsorbed to a column of DEAE ion exchange chromatography and eluted with a salt gradient. The factor was heat-labile and trypsin-sensitive. The activity was not destroyed by a reducing agent including dithiothreitol. This factor stimulated the proliferation of vascular endothelial cells but was found to be inactive against normal rat kidney fibroblasts.     

Purification and Properties of the Trypsin Inhibitors from Buckwheat Seed

The trypsin inhibitors in buckwheat seeds were isolated by affinity chromatography on trypsin-Sepharose 4B, and the components were fractionated by chromatography on DEAE-Sepharose CL-6B. The major components, inhibitors I, II and III, were found to be homogeneous proteins with molecular weight of about 8,000. Trypsin inhibitory activity was more pronounced than the chymotrypsin inhibitory activity in all the inhibitor preparation obtained. The three major inhibitors had similar amino acid compositions and had no detectable amounts of tryptophan and carbohydrate. A high level of acidic and basic amino acid residues and a low level of methionine, tyrosine and phenylalanine residues characterized the inhibitors. Although the inhibitors I and II were particularly thermostable, inhibitor III, the most abundant component, was shown to be relatively heat-labile.     

Partial purification and properties of long-chain acyl-CoA hydrolase from rat brain cytosol

Long-chain acyl-CoA hydrolase (EC 3.1.2.2.) has been partially purified from the 100,000 × g supernatant fraction of rat brain tissue. The purification procedure included chromatography on gel filtration media, DEAE-cellulose, CM-cellulose, and hydroxyapatite. The partially purified enzyme had a specific activity of 7.1 mol/min-mg, and when analyzed by polyacrylamide gel electrophoresis, revealed one major and three minor bands of protein in the presence of dodecyl sulfate and two major bands of protein in the absence of dodecyl sulfate. The enzyme had a molecular weight of 65,000 and showed no evidence of aggregated or dissociated forms. The highest catalytic activity was exhibited with palmitoyl-CoA and oleoyl-CoA as substrates. Lower activity was found with decanoyl-CoA as the substrate and little or no activity was found with acetyl-CoA, malonyl-CoA, butyryl-CoA, or acetoacetyl-CoA. The enzyme was inhibited by CoA, various metal ions, including Mn²⁺, Mg²⁺ and Ca²⁺, and by bovine serum albumin. Heating the enzyme produced a loss of activity which corresponded to a first-order kinetic process, the rate of which was independent of the choice of substrate used to measure enzyme activity. This finding supports the idea that the purification procedure yields a single species of long-chain acyl-CoA hydrolase.     

Purification and Characterization of the Heat-Stable Factors Essential for the Conversion of Lignoceric Acid to Cerebronic Acid and Glutamic Acid: Identification of N-Acetyl-l-Aspartic Acid

Abstract: The conversion of lignoceric acid to cerebronic acid, ceramides, cerebrosides, and glutamic acid is catalyzed by a rat brain particulate preparation. The heat-stable factor, prepared from calf cerebellum, together with the heat-labile factor, a pyridine nucleotide, and Mg²⁺ are essential to all of these metabolic pathways. Our previous work showed that the heat-stable factor is composed of at least two components, HSF-1 and HSF-2, and identified HSF-2 as d -glucose-6-phosphate. In the current investigation, HSF-1 was further purified and found to be N -acetyl- l -aspartic acid. In addition, it was discovered that a third component, HSF-3, is also required for heat-stable factor activity. A reconstituted system composed of N -acetylaspartic acid, glucose-6-phosphate, and HSF-3 fully replaced the heat-stable factor essential for the conversion of lignoceric acid to cerebronic acid and glutamic acid. The reconstituted heat-stable factor did not show the initial time lag always observed with the crude heat-stable factor.     

α-Hydroxylation and oxidation of lignoceric acid in brain: The role of heat-stable and heat-labile factors

Our previous investigations disclosed that the heat-stable and heat-labile factors obtained from brain cytosol are required for -hydroxylation and oxidation of lignoceric acid by rat brain particulate fraction. The heat-stable factor was recently found to contain glucose-6-phosphate, N-acetylaspartate, glutamate, aspartate, glutamine, inorganic phosphate and low levels of adenosine nucleotide as active components. A combination of these compounds was as effective as the crude heat-stable factor for enzymic activity. Using these compounds, we reinvestigated the requirement for the heat-labile factor. With crude heat-stable factor there was an absolute requirement for the heat-labile factor; however, with various combinations of the individual components of the heat-stable factor, some degree of activity was obtained without the heat-labile factor. When aspartate or one of its derivatives, N-acetylaspartate or oxaloacetate, was used in place of the heat-stable factor, the activity was relatively low but highly stimulated by the addition of heat-labile factor. On the other hand, higher activity was obtained when glutamate or one of its derivatives, glutamine or -ketoglutarate, was used without heat-labile factor. The addition of heat-labile factor to this system did not stimulate the activity. When studying the aspartate family, we discovered that the requirement for the heat-labile factor varied in a descending order: N-acetylaspartate > aspartate > oxaloacetate. Lignoceric acid oxidation was further characterized with rat brain particulate fraction, NADPH, Mg²⁺, glutamate, inorganic phosphate, and AMP without heat-stable and heat-labile factors. It was found that the requirement for NADPH was also partially eliminated with glutamate but not aspartate. The effects of various inhibitors, such as inhibitors of the electron transfer system, oxidative phosphorylation, the enzymes involved in citric acid cycle, and glycolysis, suggest that the heat-stable factor is involved in producing ATP or other high energy compounds to be used for the activation of lignoceric acid. ATP added to the system in place of heat-stable factor resulted in less than one-half of the lignoceric acid oxidation.