Spectrophotometric determination of lipases, lysophospholipases, and phospholipases

Spectrophotometric techniques for determining the activities of lipases, lysophospholipases, and phospholipases are reviewed. These methods involve the use of thioester substrate analogs as well as omega-nitrophenyl derivatives of the corresponding lipids. The most promising results are obtained with the thioester substrate analogs. Mono- and diacylglycerol lipases are assayed by using rac-1-S-decanoyl-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-1-mercapto-2,3-propanediol, respectively. Phospholipases A1 and A2 are determined by using rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and 2-hexadecanoylthio-1-ethyl-phosphocholine, respectively. Lysophospholipases are measured by using 2-hexadecanoylthio-1-ethyl-phosphocholine. Phospholipase C is assayed with rac-1-S-phosphocholine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol. Thioester substrate analog assay procedures are more rapid, sensitive, convenient, continuous, and less expensive than the classical radiochemical techniques.     

Hydrolysis of thioester analogs by rat liver phospholipase A1

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4'-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.     

Cyclosporin A inhibits the proliferative response and the generation of helper, suppressor and cytotoxic T-cell functions in the autologous mixed lymphocyte reaction

Chemical oxidation or reduction of lymphocyte cell surface thiol or disulfide groups, respectively, has been shown to alter the proliferative activity of murine T cells. S-2-(3-aminopropylamino)ethylphosphothioic acid, a compound containing no free thiol group until it is intracellularly dephosphorylated, did not enhance Con A-induced proliferation which suggested that thiols did not mediate proliferative enhancement via an intracellular mechanism. Glutathione, an impermeant thiol, enhanced T-cell proliferation 68% as effectively as 2-mercaptoethanol (2-ME), which suggested that the thiol-sensitive site was at the cell surface. A battery of structural analogs to 2-ME was employed to elucidate the chemical requirements for the biological activity of the thiols. The necessity for a hydrogen-binding moiety on the thiol reagent was determined by the use of non-hydrogen-binding analogs and by competitive inhibition of the thiol-enhancing activity of 2-ME by non-thiol-containing hydrogen-binding analogs. Pretreatment of cells with the copper:phenanthroline complex (CuP), an impermeant oxidant of thiol groups, reduced the Con A-induced response >79%; however, the presence of 2-ME in culture completely reversed the inhibitory effect of CuP pretreatment. Oxidation of T cells by high oxygen tension (17% O₂) also ablated the Con A response but did not alter the response to Con A + 2-ME. Protection from oxidative inhibition also was afforded T cells by sequential reduction and blockage of cell surface thiol groups. Finally, a model which correlates the chemical study of cell surface residues with T-lymphocyte responsiveness is presented.     

Mn2+ does not uncouple adenosine "Ra" receptors from the liver adenylate cyclase

Four analogs of oxymorphone, oxymorphaminoethylthiol, oxymorphamino-ethyldisulfide, oxymorphaminoethyl-nitrobenzoic acid disulfide and oxymorphone thiazolidine, as well as the enkephalin analogs, enkephalin-thiol, Tyr-D-Ala-Gly-Phe-Leu-Lys(?-NH)COCH₂CH₂SH and the enkephalin-dimer, [Tyr-D-Ala-Gly-Phe-Leu-Lys(?-NH)COCH₂CH₂S-]₂, were examined for binding to enkephalin and morphine receptors. The analogs gained substantial affinity for enkephalin and lost affinity for morphine receptors. The affinity of the dimers of both opiates and enkephalins was slightly greater than that achieved by the corresponding thiol monomers. However, in the guinea pig ileum the dimeric analogs were much more active than the monomers. Receptor dimerization or cross-linking may be involved in the biological activity of opiates and opioid peptides.     

Interaction of S-activated enkephalin analogs with opiate receptors

Enkephalin analogs containing a thiol activated by a thiomethyl (SCH3)*** or 3-nitro-2-pyridinesulfenyl (Npys) group were synthesized. Incubation of such S-activated enkephalin analogs as [D-Ala2, Leu(CH2S)SCH(3)5]enkephalin or [D-Ala2,Leu(CH2S)Npys5]enkephalin with guinea pig ileum (GPI) resulted in the continuous stimulation of the mu opiate receptors. This sustained GPI-activity was completely reversed with the antagonist naloxone, while subsequent washings elicited again the full enkephalin activity. When GPI showing full enkephalin activity was incubated with 1 mM dithiothreitol, about 70% of the activity was eliminated. Examination of enkephalin analogs containing Cys(Npys) at position 1, 5, or 6 suggested that no other thiols occur near the enkephalin binding site of the mu receptor. From these results, it is considered that only one thiol group exists near the binding site of the mu receptor in GPI. Similar results were also obtained for the mu receptors in mouse vas deferens.     

Chiral synthesis of a dithiolester analog of phosphatidylcholine as a substrate for the assay of phospholipase A2

The synthesis of a dithiolester analog of phosphatidylcholine, 1,2-bis(heptanoylthio)-1,2-dideoxy-sn-glycerol-3-phosphocholine (thio PC), is described. Starting with 1-trityl-sn-glycerol (prepared from D-mannitol), tosylation followed by displacement with potassium methyl xanthate gave a trithiocarbonate. Reductive cleavage of the latter gave a 1,2-dithiol which was then acylated, detritylated, and esterified with choline phosphate. Hydrolysis of thio PC by phospholipase A2 (Naja naja) indicated 95% chiral purity. The rate of hydrolysis as a function of substrate concentration showed a sharp increase at about 0.17 mM, the critical micellar concentration of the lipid. A spectrophotometric assay of phospholipase A2 (by measurement of released thiol groups in the presence of dithionitrobenzoic acid) was quite sensitive. As little as 1 ng of enzyme was detected, representing a rate of about 0.2 nmol of substrate hydrolyzed per min.     

Bacillus cereus phospholipase C: carboxylic acid ester specificity and stereoselectivity

Thiophosphate analogs of phosphatidylcholine have been synthesized with varying structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C (Bacillus cereus) to estimate the minimal structural requirements associated with the non-polar portion of the substrate phospholipid. The analogs were of three types containing zero, one or two carboxylic acid ester functionalities. The analogs with one or two ester groups acted as substrates for phospholipase C, while those without an ester functionality were not hydrolyzed. The rac-phosphatidylcholine analog with two ester functionalities gave biphasic time-course results, and was subsequently resolved into enantiomers by selective hydrolysis with a sterospecific phospholipase A2 (Crotalus atrox). The enantiomer with R absolute configuration was rapidly hydrolyzed by the phospholipase C while the enantiomer with the S configuration was slowly hydrolyzed after a long induction period. The results suggest that the B. cereus phospholipase C is specific for an ester functionality and is stereoselective for the R absolute configuration at glycerol C-2.     

Spectrophotometric measurement of mercaptans with 4,4'-dithiodipyridine

In a preceding study, 4,4'-dithiodipyridine (DTDP) was shown to be superior to 5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent) for spectrophotometric measurement of thiol groups in aqueous solution. (i) Sensitivity is higher because a larger absorbance increase is seen at a given thiol concentration. (ii) The intrinsic reactivity of thiolate anions for DTDP is much higher than for Ellman's reagent; thus, the reaction can be carried out at pH > or = 4.5 instead of at pH 8.0. In the present study, these advantages of DTDP were exploited for spectrophotometric measurement of thiols in organic solvent. DTDP was found to quantitatively react with nonpolar thiols when triethylamine was used as catalyst, intense light absorption (between 344 and 360 nm) was seen when the reaction was terminated with acetic acid, and the spectrophotometric responses were independent of the nonthiol portions of the mercaptans. The determination limit (10x the standard deviation of the reagent blank) was 3 microM, and the upper limit was approximately 40 microM on a typical spectrophotometer. The thiol contents of the mercaptans were independently verified by a modification of standard iodometry in which toluene/butanol or chloroform/butanol was included to dissolve nonpolar mercaptans.     

The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine

Thiophosphate containing analogs of phosphatidylcholine have been synthesized with varying degrees of structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C from Clostridium perfringens in order to examine the requirement for substrate ester functionalities and the stereoselectivity of the enzyme. The substrate analogs with ester groups in the nonpolar portion of the molecule were acceptable substrates for phospholipase C, while those analogs without ester functionalities were not hydrolyzed. Substrate analogs with chiral centers were resolved using the stereospecificity of phospholipase A2 from Crotalus atrox venom. These resolved substrates were used to study the biphasic hydrolytic time courses observed when rac-dioctanoylphosphatidylthiocholine was used as substrate. The "naturally occurring" enantiomer with R absolute configuration was rapidly hydrolyzed in the presence of phospholipase C while the "nonnaturally occurring" enantiomer with S configuration was slowly hydrolyzed only after a long induction or "lag" period. The selectivity for the R enantiomer over the S enantiomer can be lessened by altering the composition of the substrate micelles resulting in accelerated rates of hydrolysis of the S enantiomer.     

1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine, a highly specific chromogenic substrate of phospholipase A2

1-Palmitoyl-2-thiopalmitoyl phosphatidylcholine (2-thioPC), a structurally modified phospholipid analog is specifically hydrolyzed by phospholipase A2 to liberate 2-thiolysophosphatidylcholine and palmitic acid. The sulfhydryl group of the product is readily trapped by 5,5'-dithiobis (2-nitrobenzoic acid) allowing continuous spectrophotometric monitoring of the enzymatic reaction. The rates of hydrolysis by bee-venom phospholipase A2 have been determined in a series of Triton X-100 containing mixed micelles. At 1 mM 2-thioPC increasing the concentration of Triton X-100 from 4 to 16 mM changes the specific activity of bee-venom phospholipase A2 from 96.9 to 17.9 mumol/min/mg, about one order of magnitude lower than dipalmitoyl phosphatidylcholine hydrolysis in micelles of similar composition. The chromogenic substrate is the first phospholipid analog exhibiting absolute specificity for phospholipase A2 and should be applicable to spectrophotometric detection and kinetic characterization of both water soluble and membrane-bound forms.     

The comparative structure of mammalian glyceraldehyde 3-phosphate dehydrogenase

1. The amino acid sequences around the thiol groups of glyceraldehyde 3-phosphate dehydrogenase from badger and monkey skeletal muscle were compared with the sequences around the thiol groups in the enzyme isolated from other organisms. 2. Preliminary evidence of the existence of isoenzymes in the badger was obtained. Only the major form, however, could be purified completely. 3. The monkey enzyme contains only three cysteine residues per polypeptide chain compared with the four found in all the other mammalian enzymes so far examined, including that of badger, and the two in yeast. The missing thiol group in monkey was identified as residue 281 in the corresponding sequence of the pig enzyme. 4. These experiments rule out any essential role for cysteine-281 in the function of the mammalian enzymes. 5. Further evidence of the remarkable conservation of amino acid sequence in this enzyme during evolution is presented and discussed.     

Studies on lysophospholipases, V. The action of lysolecithin-hydrolyzing enzymes on lecithins and 1-acyl lysolecithins with varying fatty acid chain-length.

The activity of two purified lysolecithin-hydrolyzing enzymes on homologous series of synthetic lecithins containing two identical fatty acyl chains and of 1-acyl-lysolecithins has been measured as a function of substrate concentration. In general, enzymatic activity toward lecithins decreased with increasing chain length. Maximal hydrolysis rates for the lysolecithin series were measured with 1-dodecanoyllysolecithin. In this series increased affinities for substrates with increasing acyl-chain length was noticed. In the substrate concentration versus enzymatic velocity curves no breaks were observed at the critical micelle concentration of the various substrates. The initial site of attack during hydrolysis of short-chain lecithins was determined using 1-octanoyl-2pentanoyl-lecithin, 1-hexanoyl-2-hexyllecithin and 1 -hexyl-2-hexanoyllecithin. Both enzymes exhibited a pronounced preference for hydrolysis of the acyl ester bond at the 1-position. Especially the enzyme from beef pancreas seems to be suitable for the enzymatic preparation of 2-acyl lysolecithins from the corresponding short-chain lecithins.     

Folate-dependent hydrolysis of acetyl-coenzyme A by recombinant human and rodent arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) are drug and xenobiotic metabolizing enzymes that catalyze the N-acetylation of arylamines and hydrazines and the O-acetylation of N-hydroxy-arylamines. Recently, studies report that human NAT1 and mouse Nat2 hydrolyze acetyl-coenzyme A (AcCoA) into acetate and coenzyme A in a folate-dependent fashion, a previously unknown function. In this study, our goal was to confirm these findings and determine the apparent Michaelis–Menten kinetic constants (V_max and K_m) of the folate-dependent AcCoA hydrolysis for human NAT1/NAT2, and the rodent analogs rat Nat1/Nat2, mouse Nat1/Nat2, and hamster Nat1/Nat2. We also compared apparent V_max values for AcCoA hydrolysis and N-acetylation of the substrate para-aminobenzoic acid (PABA). Human NAT1 and its rodent analogs rat Nat2, mouse Nat2 and hamster Nat2 catalyzed AcCoA hydrolysis in a folate-dependent manner. Rates of AcCoA hydrolysis were between 0.25–1% of the rates for N-acetylation of PABA catalyzed by human NAT1 and its rodent orthologs. In contrast to human NAT1, human NAT2 and its rodent analogs rat Nat1, mouse Nat1, and hamster Nat1 did not hydrolyze AcCoA in a folate-dependent manner. These results are consistent with the possibility that human NAT1 and its rodent analogs regulate endogenous AcCoA levels.     

Disruption of Potential α-Helix in the G Loop of the Guinea: Pig 5-Hydroxytryptamine2 Receptor Does Not Prevent Receptor Coupling to Phosphoinositide Hydrolysis

Abstract: Heterogeneity of the 5-hydroxytryptamine₂ (5-HT₂) receptor across species has been implicated in several pharmacological and physiological studies. Although 5-HT₂ receptors in the rat have been linked to increases in Phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5-HT₂ receptors with Pl hydrolysis, the second messenger generally linked with 5-HT₂receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5-HT₂ receptors exist between rat and guinea pig. First, we isolated partial cortical 5-HT_a receptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor-effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5-HT₂ receptor were 97% homologous. However, the guinea pig 5-HT₂ receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5-HT₂ receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5-HT₂ receptor from guinea pig. 5-HT and the 5-HT₂ receptor agonist α-methyl-5-HT increased PI hydrolysis in guinea pig cortical slices whereas the 5-HT_1c receptor agonist 5-methyltryptamine was significantly less potent. In addition, the 5-HT₂ receptor antagonists LY53857, ketanserin, and spiperone blocked 5-HT-stimulated Pl hydrolysis. These pharmacological data suggested that activation of the 5-HT₂ receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5-HT₂ receptor that influence receptor-effector coupling were different from the rat, such differences were not critical to receptor-effector coupling because, as in the rat, guinea pig brain 5-HT₂ receptors were also coupled to PI hydrolysis.     

A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase

A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H⁺ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88–95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of K₁ for edrophonium, decamethonium and Al³⁺.     

Ultrastructural evidence for uptake of silicon-containing silicic acid analogs byUrtica pilulifera and incorporation into cell wall silica

Summary In natural environments the stinging nettle plant,Urtica pilulifera, bears stinging cells in which electron dense silica deposits occupy a significant volume of the cell wall. Plants were grown in hydroponic solutions with and without supplements of silicic acid, the chemical form of silicon available to biological systems to determine if this plant and the stinging cells will grow normally under conditions of silicon starvation. In separate experiments, several analogs of silicic acid were added as supplements to the hydroponic solution to determine whether silicic acid binding sites had detectably different specificities for the different molecular structures of the analogs. The analogs [(R-)_nSi(-OH)_m] have the following structures (R, n, m): (1)-H, 1, 3; (2)-CH₃, 1, 3; (3)-CH₃, 2, 2; (4)-CH₃, 3, 1; (5)-CH₂CH₃, 1, 3; and (6)-C₆H₅, 1, 3. Electron microscopy was used as an assay for the uptake and incorporation of analogs into an electron dense silica-like product in the stinging cell wall. The results indicate that cell wall silica production occurred only when the analog contained at least three hydroxyl groups. The morphology and ontogeny of the plant was normal except for: 1, the appearance of green spots on the leaves when the analog contained two or more hydroxyl groups, and 2, total blockage of flowering by the two methyl derivative of silicic acid, (CH₃)₂Si(OH)₂.     

Retinoic acid-binding protein: a plasma membrane component

Soluble protein extracts from lyophilized plasma membranes prepared from chick embryo skin and transplantable murine carcinomas were found to contain a specific retinoic acid-binding component. This binding component showed high affinity for biologically active analogs of retinoic acid. The plasma membrane component exhibited similar physicochemical properties to those of the retinoic acid-binding protein described earlier in the cytosol. The protein exhibited mercurial-sensitive thiol functions in ligand binding; the mercurial-inhibition was reversed on treatment with thiol compounds. The plasma membrane binding component may be involved in the cellular uptake of retinoic acid.     

Effect of modification of enkephalin C-terminal functions on affinity selection of opioid receptors

Enkephalin analogs with various C-terminal modifications have been synthesized to evaluate the corresponding structural elements in the opioid receptors. The carboxyl group of the C-terminal leucine5 or glycine5 was converted into the mercaptomethyl (-CH2SH) and hydroxymethyl (-CH2OH) groups, starting from leucinthiol or leucinol for Leu5-derivatives and from cysteamine or ethanolamine for Gly5-derivatives. Interactions of synthetic peptides with the opioid receptors were examined by the radioligand receptor binding assays using rat brain and tritiated enkephalin analogs. The data suggest that the C-terminal carboxyl group in enkephalins is important, but not electrostatically, for interaction with delta-opioid receptors. With leucinthiol-enkephalin in biological assays which examine its inhibitory activity for electrically stimulated contractions of isolated smooth muscle, it was found that the reactive thiol group exists in the mu receptors present in the guinea pig ileum. Leucinthiol-enkephalin became bound covalently to this receptor-thiol group via disulfide formation after prolonged incubation.     

Cathepsin L--a latent proteinase in guinea pig sperm

Guinea pig spermatozoa were found to contain a fully-latent cysteine proteinase that could be unmasked by incubating epididymal sperm for 2 hr at pH 3.5 and 37 degrees C. The proteinase was identified as cathepsin L (EC 3.4.22.15) on the basis of its optimal hydrolysis of benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide (Z-Phe-Arg-NMec) at pH 5.5; lack of action on Z-Arg-Arg-NMec and Arg-NMec; urea-enhanced digestion of azocasein; marked sensitivity to thiol reagents, leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxy-succinylleucylamido(3-methyl)butane (Ep-475 or E-64-c); and insensitivity to pepstatin and serine proteinase inhibitors. Gossypol, a male antifertility agent, was inhibitory. The unmasking phenomenon was reversibly inhibited by HgCl2 and mersalyl acid, and prevented by leupeptin and Ep-475, but not by pepstatin.     

Regioselective Enzymatic Hydrolysis of 3′,5′-DI-O-Acetylthymidine and Observation of a Surface Effect Due to Polystyrene

The selective enzymatic hydrolysis of 3′,5′-di-O-acetylthyidine (1) was studied. The lipases from porcine pancreas and Aspergillus niger, and pig liver esterase, all catalysed selective hydrolysis of the 5′O-acetyl group, but the lipase from Candida cylindracea catalysed selective hydrolysis of the 3′-O-acetyl group. Highest selectivity, leading to essentially pure 3′-O-acetylthymidine, was achieved using porcine pancreatic lipase in dilute solution at pH 7.5. Provision of an artificial interface in the form of polystyrene beads led to a significant increase in the rate of hydrolysis, accompanied by a marked fall in selectivity. Other changes in the hydrolysis conditions, such as raising the concentration of substrate or adding cosolvent, also led to a fall in selectivity.