Lysophosphatidylcholine acyltransferase and lysophosphatidylcholine: lysophosphatidylcholine acyltransferase in alveolar type II cells from fetal rat lung

The specific activity of lysophosphatidylcholine acyltransferase in sonicated fetal rat lung type II cells was found to be an order of magnitude greater than that of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase. The specific activity of lysophosphatidylcholine acyltransferase in sonicated fetal rat lung type II cells increases towards the end of gestation, whereas that of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase does not show a change. While lysophosphatidylcholine acyltransferase in whole fetal lung homogenate is more active towards oleoyl-CoA than towards palmitoyl-CoA, the enzyme in sonicated fetal type II cells is more active towards palmitoyl-CoA. If measured with palmitoyl-CoA as acyl donor, the specific activity of lysophosphatidylcholine acyltransferase in type II cells is higher than that in whole lung during late gestation. In contrast, the specific activity of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase in type II cells is lower than that in whole lung. These observations indicate that in fetal rat type II cells the deacylation-reacylation cycle is more important for the formation of dipalmitoylphosphatidylcholine than the deacylation-transacylation process.     

Lysophospholipase and transacylase activities of rat gastric mucosa.

A transacylase that converts 1-palmitoyl lysophosphatidylcholine to dipalmitoyl phosphatidylcholine was demonstrated in the rat gastric mucosa. This enzyme required neither ATP or CoA nor bile salt and detergent for its activity. The enzyme preparation also exhibited powerful lysophospholipase activity. The transacylase and lysophospholipase were both located in the cytosol fraction, and their activities remained associated at a constant ratio throughout the purification steps, including the isoelectrofocusing procedure. They responded similarly with respect to the addition of metal ions, bile salt, detergent, and heat treatment. Both enzyme activities also exhibited similar apparent K_m values for lysophosphatidylcholine. These observations suggest that both the lysophospholipase and transacylase activities may reside in the same enzyme.     

Substrate specificity of lysophospholipase-transacylase from rat lung and its action on various physical forms of lysophosphatidylcholine

Lysophospholipase-transacylase (lysolecithin acylhydrolase, EC 3.1.1.5) from rat lung catalyzes the transfer of acyl groups from lysophosphatidylcholine to either water or another molecule of lysophosphatidylcholine. Studies on the substrate specificity of the purified enzyme showed that a phosphate group in the substrate is essential for enzymatic activity; monoacylglycerol is not hydrolyzed, nor does it serve as an acceptor of acyl groups. The influence of the acyl chain in lysophosphatidylcholine was investigated by using mixtures of differently labelled lysophosphatidylcholine species, or by studying the transfer of [1-¹⁴C]Palmitate from [1-¹⁴C]palmitoylpropane (1,3)diol-phosphocholine to various 1-acyl-sn-glycero-3-phosphocholines. Lysophosphatidylcholines with acyl chains comprised of ten or more C-atoms were found to serve as acyl acceptors. This finding was used to determine the action of the enzyme on 1-[1-¹⁴C]lauroyl- and 1[1-¹⁴C]myristoyl-sn-glycero-3-phosphocholine both below and above the critical micelle concentration of the substrate. Monomeric substrate was effectively hydrolyzed, but the transacylase activity of the enzyme was only expressed when substrate micelles were present. Likewise, no transacylase activity was found when lysophosphatidylcholine was embedded in liposomal membranes prepared from lung total lipids. These findings, which persist with crude enzyme preparations (100 000 × g supernatant), are discussed in relation to the putative function of the lysophospholipase-transacylase in the synthesis of disaturated phosphatidylcholine in lung.     

Substrate selectivity of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung

The influence of both polar group and acyl chain of lysophospholipids on the lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung was studied. Both, transacylase and hydrolase activities of this enzyme, utilize selectively 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine when compared with 1-[9,10-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine. Transacylase activity is more selective for lysophosphatidylcholine as acyl acceptor than as acyl donor. The amount of dipalmitoylphosphatidylcholine/min/mg protein synthesized from mixed lysophosphatidylcholine/lysophosphatidylethanolamine micelles does not change with increasing molar percentages of lysophosphatidylethanolamine in the mixture and is similar to that formed with pure lysophosphatidylcholine micelles. Transacylation reaction takes place preferentially with long and saturated acyl chains whereas hydrolysis reaction does more efficiently with longer acyl chains, independently of their insaturation degree.     

Evidence for a single rat thyrotropin-beta-subunit gene: thyroidectomy increases its mRNA

We have isolated and characterized cDNAs representing the rat thyrotropin-beta-subunit. The cDNAs were prepared from poly(A)+ RNA obtained from rat pituitary glands and encode the precursor of the rat thyrotropin-beta-subunit which contains a leader or signal peptide of 20 amino acids, and an apoprotein of 118 amino acids. Blot hybridization analysis of total rat liver DNA digested with several restriction enzymes indicates the likelihood of a single gene encoding the rat thyrotropin-beta-subunit. In addition, analyses of pituitary RNA from normal and thyroidectomized rats indicate that the mRNA encoding the rat thyrotropin-beta-subunit is approximately 700 bases in length and its level increases 8--10-fold after thyroid gland ablation.     

Substrate specificity of a CoA-dependent stearoyl transacylase from bovine testis membranes.

We identified a CoA-dependent stearoyl transacylase activity in bovine testis membranes, then examined the enzyme's specificity in mixed micelle systems containing the neutral detergent Triton X-100. The enzyme transferred stearoyl groups from a variety of phospholipids to sn-2-arachidonoyl lysophosphatidic acid (lysoPA), but showed very little palmitoyl transacylase activity. Its ability to transfer stearoyl groups was both donor- and acceptor-dependent. For example, it used weakly acidic phospholipids, such as sn-1-stearoyl-2-acyl species of phosphatidylinositol (PI), as donors, but did not use phosphatidylinositol-4,5-bisphosphate or sn-1-stearoyl-2-arachidonoyl phosphatidylcholine. Moreover, it used sn-2-acyl species of lysoPA and sn-2-arachidonoyl lysoPI as acceptors but did not use sn-2-arachidonoyl species of lysophosphatidylserine, lysophosphatidylethanolamine, or lysophosphatidylcholine. When taken together, our results raise the possibility that sn-1-stearoyl-2-acyl species of PI may be the primary acyl donors in the transacylase reaction in vivo, while sn-2-acyl species of lysoPA may be the primary acyl acceptors. Available evidence suggests that the PA that is formed may subsequently be converted into PI, but the metabolic fate of the other reaction product, sn-2-acyl lysoPI, remains to be determined.     

An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine

Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 μl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.     

Evidence for the existence of a single enzyme catalyzing the phosphorylation of choline and ethanolamine in primate lung.

Choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) has been isolated and purified 1000-fold from adult African Green monkey lung with a yield of 10%. The purified enzyme also phosphorylated ethanolamine (ratio of ethanolamine kinase to choline kinase = 0.30). This ratio remained constant throughout the purification procedure. The Km for choline (3.0 - 10(-5) M) was lower than that of ethanolamine (1.2 - 10(-3) M.) Choline was also found to inhibit ethanolamine kinase activity by 50% at a concentration of 0.005 mM, while ethanolamine inhibited choline only at very high concentrations (100--150 mM). When the enzyme was subjected to inactivation by heat, hemicholinium-3, trypsin digestion, and p-hydroxymercuribenzoate, both ethanolamine kinase and choline kinase activities were destroyed at the same rate. Freezing and thawing in the absence of glycerol also destroyed both activities at the same rate. Based on these findings, we conclude that in adult African Green monkey lung tissue, there is only one enzyme for the phosphorylation of ethanolamine and choline, and that choline phosphorylation predominates.     

Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.     

Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung.

A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy. Also, kinetics of inhibition by products behaved as expected. It was concluded that the competition between two nucleophiles for the covalent acyl-enzyme intermediate, and not a different enzyme action depending on the physical state of the substrate, is responsible for the differences in kinetic pattern for the two activities of the enzyme. This conclusion, together with the fact that the kinetic equation for the transacylation is quadratic, generates a 'hysteretic' pattern that can provide the basis of self-regulatory properties for enzymes to which this model could be applied.     

Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver.

Glutathione $\text{S}$-transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on $\text{S}$-hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.     

Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines.

The enzymic 5'-deiodination of 3',5'-di-iodothyronine and 5-deiodination of 3,3',5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3',5'-Di-iodothyronine was a competitive inhibitor of 3,3',5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3',5-tri-iodothyronine was a competitive inhibitor of 3',5'-di-iodothyronine 5'-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.     

Hydrolysis of substance P and its analogs by angiotensin-converting enzyme from rat lung. Characterization of endopeptidase activity of the enzyme

Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.     

Acyl esters of polyprenols: specificity of microsomal transacylase for polyprenols of different chain length and saturation

Transfer of fatty acids from phospholipids to polyprenols, catalysed by the transacylase from rat liver microsomes, was investigated. The specificity of the enzyme for polyprenols of different chain length and different degree of saturation was studied using individual isoprenologues, the preparation of which in highly tritiated form is described. It was found that short-chain polyprenols are better substrates for the enzyme than long-chain polyprenols, and alpha-saturated better than unsaturated or multiply saturated polyprenols. Short-chain, alpha-saturated single isoprenologues were several-fold more active as acyl acceptors than natural dolichol.     

Glucose-6-phosphate dehydrogenase from a tetracycline producing strain of Streptomyces aureofaciens: some properties and regulatory aspects of the enzyme

Glucose-6-phosphate dehydrogenase from Streptomyces aureofaciens exhibited activity with both NAD and NADP, the maximum reaction rate being 1.6 times higher for NAD-linked activity than for the NADP-linked one. The KM values for NAD-linked activity were 2.5 mM for glucose-6-phosphate and 0.27 mM for NAD, and for NADP-linked activity 0.8 mM for glucose-6-phosphate and 0.08 mM for NADP. NAD- and NADP-linked activities were inhibited by both NADH and NADPH. (2'-phospho-)adenosinediphospho-ribose inhibited only NAD-linked activity. The inhibition was competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate.     

Hatching in the pike Esox lucius L.: Evidence for a single hatching enzyme and its immunocytochemical localization in specialized hatching gland cells

Antibodies against purified hatching enzyme (HE) from the pike, Esox lucius L., have been used to examine different aspects of the presence of the enzyme in the ontogeny of this teleostean fish. Immunochemical analysis indicates that the two proteolytic enzymes which occur in the hatching medium arise from a single protease, HE itself. The second proteolytic fraction found in gel filtration of hatching medium could be a heterogeneous population of complexes of HE with digestion fragments of its natural substrate, the zona radiata. Immunofluorescence microscopy by means of anti-HE antibodies demonstrates that HE is localized in the so-called hatching gland cells (HGCs). The HGCs in pike appear as oval to round cells 10–15 μm in diameter containing granules of 1.5–2.3 μm. They are found interspersed between the periderm and the presumptive epidermis. The number of HGCs and their granule content increase significantly until the 35-somite stage to reach about 1200 and 30, respectively. From then on these numbers do not change until hatching in the 66-somite stage. The distribution of the HGCs over the embryo also changes, probably since HGC precursors in the yolk sac differentiate to HGCs later than their counterparts in the head region. The immunocytochemical procedure further shows that HE can be detected from the 10-somite stage on. Discrete hatching gland remnant bodies, phagocytized by epidermal cells, are observed in larval stages until 3–7 days after emergence of the embryo.     

Glutamate dehydrogenase: some properties of the rat brain enzyme from different cellular compartments

1. Differences in the GDH activity of neuronal, glial cells and synaptosomes were detected. 2. The enzyme was measured in both directions: synthesis and degradation of glutamate. 3. Synaptosomes were the region with the highest GDH activity. 4. ADP plays an important role in the regulation of the reaction sense. 5. This effector produced higher activation on the enzyme measured in the direction of glutamate synthesis than in the sense of its degradation. 6. The enhancement produced by ADP was dependent on the enzyme localization. The ADP effect is discussed.     

Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2

A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50 degrees C. The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60 degrees C and 65 degrees C for 30 min, respectively. Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-beta-mercaptoethanol and DFP. The protease from B. subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile.