Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro

Concomitant hydroxylation of proline and lysine residues in protocollagen was studied using purified enzymes. The data suggest that prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) and lysyl hydroxylase (peptidyllysine, 2-oxoglutarate; oxygen 5-oxidoreductase, EC 1.14.11.4) are competing for the protocollagen substrate, this competition resulting in an inhibition of the lysyl hydroxylase but not of the prolyl 4-hydroxylase reaction. When the same protocollagen was used for these hydroxylases, the affinity of prolyl 4-hydroxylase to the protocollagen substrate was about 2-fold higher than that of lysyl hydroxylase. Hydroxylation of lysine residues in protocollagen had no effect on the affinity of prolyl 4-hydroxylase, whereas hydroxylation of proline residues decreased the affinity of lysyl hydroxylase to one-half of the value determined before the hydroxylation. When enzyme preparations containing different ratios of lysyl hydroxylase activity to prolyl 4-hydroxylase activity were used to hydroxylase protocollagen substrate, it was found that in the case of a low ratio the hydroxylation of lysine residues seemed to proceed only after a short lag period. Accordingly, it seems probable that most proline residues are hydroxylated to 4-hydroxyproline residues before hydroxylation of lysine residues if the prolyl 4-hydroxylase and lysyl hydroxylase are present as free enzymes competing for the same protocollagen substrate.     

Prolyl 3-hydroxylase: partial characterization of the enzyme from rat kidney cortex.

The formation of 3-hydroxyproline was studied with crude rat kidney cortex extract as a source of enzyme and chick embryo tendon protocollagen and procollagen or cartilage protocollagen as a substrate. Synthesis of 3-hydroxyproline was observed with all these substrates and the formation of 3-hydroxyproline ranged up to seven residues per pro-alpha-chain. The highest rate of 3-hydroxylation took place at 20 degrees C and the reaction required Fe2+, O2,2-oxoglutarate and ascorbate. The formation of 3-hydroxyproline was affected by chain length and the conformation of the substrate, in that longer polypeptide chains proved better substrates, while the native triple-helical conformation of protocollagen or procollagen completely prevented the reaction. Formation of 3-hydroxyproline with tendon procollagen as a substrate was not inhibited by antiserum to prolyl 4-hydroxylase or by poly(L-proline) when these substances were used in concentrations which clearly inhibited 4-hydroxyproline formation with tendon protocollagen as a substrate. Furthermore, pure prolyl 4-hydroxylase did not synthesize any 3-hydroxyproline under conditions in which the crude rat kidney cortex enzyme would readily do so. The data thus strongly suggest that prolyl 3-hydroxylase and prolyl 4-hydroxylase are separate enzymes.     

Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism

Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO₂, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe²⁺, α-ketoglutarate, O₂ and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO₂, succinate and Fe²⁺, in which Fe²⁺ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO₂ is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO₂ and succinate or after the release of all products, including Fe²⁺, and dehydroascorbate is released before the binding of Fe²⁺. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical.Zn²⁺, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe²⁺, α-ketoglutarate, O₂ and ascorbate respectively, and epinephrine noncompetitively with regard to all cosubstrates. Apparent K_i values are given for the product and other inhibitors.     

Separation of acetanilide and its hydroxylated metabolites and quantitative determination of "acetanilide 4-hydroxylase activity" by high-pressure liquid chromatography.

A simple and very sensitive method for the separation of 4-hydroxyacetanilide, 3-hydroxyacetanilide, 2-hydroxyacetanilide, and acetanilide was developed with the use of high-pressure liquid chromagraphy. Each of these phenolic derivatives can be separated completely from acetanilide and from one another. A simple assay for “acetanilide 4-hydroxylase activity” is thus described. The limit of sensitivity for cytochrome P-450-mediated acetanilide 4-hydroxylase activity is estimated to be 1.0 pmol/min/mg microsomal protein, thereby allowing this assay to be useful in detecting monooxygenase activity in “low level” nonhepatic tissues. Hepatic acetanilide 4-hydroxylase activity is induced about fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. Although acetanilide 2-hydroxylase activity is about seven times lower than the 4-hydroxylase activity, the 2-hydroxylase is also induced about three- or fourfold in $\text{C57BL}\text{6N}$ mice by 3-methylcholanthrene. The “2-hydroxylase activity” cannot, however, be strictly quantitated under the conditions described herein. The K_m values of both the 3-methylcholanthrene-induced and control 4-hydroxylase activity are about 0.55 mm; V_max values for 3-methylcholanthrene-treated and control mice, respectively, are 4.9 ± 1.1 and 1.1 ± 0.31 nmol/min/mg microsomal protein. The 4-hydroxylase in the liver of both 3-methylcholanthrene-treated and control mice appears to represent two or more catalytic activities, i.e., two or more forms of P-450 having widely differing affinities for the substrate acetanilide.     

Assay of microsomal oxysterol 7α-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

A method of assaying hepatic cytochrome P-450, oxysterol 7α-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-³H]hydroxycholesterol as a substrate and hydroxypropyl-β-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7α,25-dihydroxycholesterol, into 3-oxo-7α,25-dihydroxy-4-cholesten by the activity of 3β-hydroxy-Δ⁵-C₂₇ steroid oxydoreductase, a microsomal NAD⁺ and NADP⁺ dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP⁺ into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7α-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (?33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (?30%) and also by the oxysterols 7α-hydroxycholesterol (?21%), 7β-hydroxycholesterol (?25%) and epicoprostanol (?20%), and by cyclosporin A (?26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.     

Inhibition of prolyl hydroxylase activity by bradykinin analogs containing a prolyl-like residue

A number of substituted bradykinin analogs were prepared in which the proline in position 3 was replaced by analogs of proline. All of the bradykinin analogs, with the exception of l-azetidine-2-carboxyl³-bradykinin showed significant ability to inhibit prolyl hydroxylase activity. Addition of an l-glutamyl residue to the amino terminus of 3,4-dehydro-l-prolyl³-bradykinin and trans-4-hydroxy-l-prolyl³-bradykinin resulted in competitive inhibitors of increased effectiveness with K_i, values approximately 10^?4m. One of the peptides, l-3,4-dehydro-l-prolyl³-bradykinin, appeared to serve as a substrate for prolyl hydroxylase.     

Separation of prolyl 3-hydroxylase and 4-hydroxylase activities and the 4-hydroxyproline requirement for synthesis of 3-hydroxyproline

Differences between prolyl 3-hydroxylase and prolyl 4-hydroxylase activities were found in their stimulation and inactivation by dithiothreitol and in their affinity to poly-L-proline linked to agarose. The two enzyme activities were separated by gel filtration, the results demonstrating that they are due to separate proteins. Comparison of [¹⁴C]proline-labelled protocollagen and the same protein when fully 4-hydroxylated as substrates indicated dependence of 3-hydroxyproline formation on the presence of 4-hydroxyproline. It is suggested that the main substrate sequence for 3-hydroxyproline synthesis is -Gly-Pro-4Hyp-Gly-.     

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2^n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2^n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.     

Prolyl 3-hydroxylase 1, enzyme characterization and identification of a novel family of enzymes

The collagen prolyl hydroxylases are enzymes that are required for proper collagen biosynthesis, folding, and assembly. They reside within the endoplasmic reticulum and belong to the group of 2-oxoglutarate and iron-dependent dioxygenases. Although prolyl 4-hydroxylase has been characterized as an alpha2beta2 tetramer in which protein disulfide isomerase is the beta subunit with two different alpha subunit isoforms, little is known about the enzyme prolyl 3-hydroxylase (P3H). It was initially characterized and shown to have an enzymatic activity distinct from that of prolyl 4-hydroxylase, but no amino acid sequences or genes were ever reported for the mammalian enzyme. Here we report the characterization of a novel prolyl 3-hydroxylase enzyme isolated from embryonic chicks. The primary structure of the enzyme, which we now call P3H1, demonstrates that P3H1 is a member of a family of prolyl 3-hydroxylases, which share the conserved residues present in the active site of prolyl 4-hydroxylase and lysyl hydroxylase. P3H1 is the chick homologue of mammalian leprecan or growth suppressor 1. Two other P3H family members are the genes previously called MLAT4 and GRCB. In this study we demonstrate prolyl 3-hydroxylase activity of the purified enzyme P3H1 on a full-length procollagen substrate. We also show it to specifically interact with denatured collagen and to exist in a tight complex with other endoplasmic reticulum-resident proteins. Immunohistochemistry with a monoclonal antibody specific for chick P3H1 localizes P3H1 specifically to tissues that express fibrillar collagens, suggesting that other P3H family members may be responsible for modifying basement membrane collagens.     

A sensitive and specific tritium release assay for dopamine-beta-hydroxylase (DbetaH) in serum.

The release of tritium from [7-³H₂]dopamine was investigated as a possible procedure for the assay for dopamine-β-hydroxylase (DβH) in rat and human serum. The release was found to have the same characteristics as those deseribed previously for DβH in serum; for example, an optimum rate of reaction at pH 5.0 or an enhancement of release with agents such as Cu²⁺ ions and N-ethylmaleimide which are known to inactivate endogenous inhibitors of DβH in serum. Tritium release was blocked by the DβH inhibitor fusaric acid but not by inhibitors of other dopamine-metabolizing enzymes in serum. Incubation of ¹⁴C-labeled dopamine along with [7-³H₂]dopamine revealed that, under the standard assay conditions, the formation of [¹⁴C]norepinephrine was accompanied by release of one of the two tritium atoms on the 7-carbon. It was concluded that the procedure provided a simple and sensitive assay of DβH activity in serum.     

Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium

O₂ regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O₂-threshold for culmination implicating it as a direct O₂-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3^SCFubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O₂-signal. Here we show that genetically induced changes in Skp1 levels also affect the O₂-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3^SCFubiquitin-ligase activity modulate O₂-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O₂-sensing.     

Inhibition of prolyl 4-hydroxylase by oxaproline tetrapeptidesin vitro and mass analysis for the enzymatic reaction products

A series of 5-oxaproline peptide derivatives was synthesized and evaluated for its ability to inhibit the prolyl 4-hydroxylasein vitro. Structure-activity studies show that the 5-oxaproline sequences, prepared by the 1,3-dipolar cycloaddition of the C-methoxycarbonyl-N-mannosyl nitrone in the presence of the ethylene, are more active than the corresponding proline derivatives. Prolyl 4-hydroxylase belongs to a family of Fe²⁺-dependent dioxygenase, which catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in-Gly-Xaa-Pro-Gly- of procollagen chains. In this paper we discover the more selective N-Cbz-Gly-Phe-Pro-Gly-OEt (K _m=520 μM) sequences which are showed stronger binding than othersin vitro. Therefore, we set out to investigate constrained tetrapeptide that was designed to mimic the proline structure of peptides for the development of prolyl 4-hydroxylase inhibitor. From this result, we found that the most potent inhibitor is N-Dansyl-Gly-Phe-5-oxaPro-Gly-OEt (K _i=1.6 μM). This has prompted attempts to develop drugs which inhibit collagen synthesis. Prolyl 4-hydroxylase would seem a particularly suitable target for antifibrotic therapy.     

Study on a prolyl endopeptidase from the skeletal muscle of common carp (Cyprinus carpio)

A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of common carp using a procedure involving ammonium sulfate fractionation and column chromatography involving DEAE-Sephacel, Phenyl-Sepharose, DEAE-Sepharose Fast Flow, and hydroxyapatite. The molecular weight of the PEP was 82 kDa as determined by SDS-PAGE. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 °C, respectively, and the K_m and k_cat were 8.33 μM and 1.71 S^?1, respectively. The activity of the PEP was inhibited by SUAM-14746, a specific inhibitor of prolyl endopeptidases, and was partially inhibited by the serine proteinase inhibitors PMSF and Pefabloc SC. According to peptide mass fingerprinting, 12 peptide fragments with a total of 134 amino acid residues were obtained, which were highly identical to prolyl endopeptidases from zebrafish (Danio rerio) and sponge (Amphimedon queenslandica), confirming the purified enzyme was a prolyl endopeptidase. Our present study for the first time reported the existence of a prolyl endopeptidase in fish muscle.     

Bioactivity of folding intermediates studied by the recovery of enzymatic activity during refolding

The peptide bond preceding proline residues realizes a cis/trans conformational switch with high switching resistance in native proteins and folding intermediates. Therefore, individual isomers have the potential to differ in bioactivity. However, information about isomer-specific bioactivities is difficult to obtain because of the risk of affecting isomeric distribution by bioactivity assay components.Here we present an approach that allows for the measurement of the recovery of enzymatic activities of wild-type RNase T₁ and RNase T₁ variants during refolding under conditions where the population of enzyme-substrate or enzyme-product complexes is negligible. Recovery of enzymatic activity was continuously monitored within the visible range of the spectrum by addition of a fluorescence-labeled nucleotide substrate to the refolding sample. We found that a nonnative trans conformation at Pro39 renders the RNase T₁ almost completely inactive. A folding intermediate having a nonnative trans conformation at Pro55 shows about 46% of the enzymatic activity referred to the native state. Pro55, in contrast to the active site located Pro39, is situated in a solvent-exposed loop region remote from active-site residues. In both cases, peptidyl prolyl cis/trans isomerases accelerate the regain of nucleolytic activity. Our findings show that even if there is a considerable distance between the site of isomerization and the active site, conformational control of the bioactivity of proteins is likely to occur, and that the surface location of prolyl bonds suffices for the control of buried active sites mediated by peptidyl prolyl cis/trans isomerases.     

A rapid and sensitive fluorescence assay for angiotensin-converting enzyme.

A new, highly sensitive, specific assay for dopamine-β-hydroxylase (DBH) activity in human serum is described. Tyramine is used as a substrate; the product of the enzymatic hydroxylation, octopamine, is converted by reacting with 1-dimethylaminonaphthalene-5-sulfonyl-chloride (Dns-Cl) to a fluorescent product, which is extracted from the reaction mixture and purified from the extract by thin-layer chromatography (tlc). The fluorescence of the dansylated octopamine is measured in situ on the tlc plate using a chromatogram-spectrofluorometer. This one-step enzyme reaction can be performed at optimum pH and substrate concentration. As little as 8 ng of octopamine can be determined accurately; the response is linear up to more than 400 ng of octopamine. A comparison with the radioenzymatic assay (Weinshilboum, R., and Axelrod, J. (1971) Circ. Res.28, 307–315) shows an approximately twofold increase in the enzymatic activity measured. Kinetic studies of human sera with high and low DBH activity gave a K_m value of 3.1 × 10^?3m. The method is successfully being used for the functional characterization of the enzyme and genetic studies (Herschel, M., in preparation).     

Radiochemical assay for renin utilizing a synthetic insoluble substrate

In order to provide a convenient in vitro assay for renin activity, a radiolabeled renin substrate analog, N-acetyl-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-His-[³H]-Leu-Leu-Val-Tyr-Ser-Gly-Lys-Pro-OH, was prepared by solid-phase synthesis. The substrate peptide was bound covalently to agarose through the ?-amino group of its lysine residue. Incubation of this insoluble complex with partially purified hog renin resulted in the release of biologically active tritiated peptide into the soluble phase of the incubation mixture, at a rate proportional to the quantity of renin added. The optimum pH for cleavage was 6.5. The apparent K_m of the substrate was 1 × 10⁻⁴M, and the V_max was 83 pmoles tritiated peptide released/min/mg renin preparation added. The minimum amount of renin detectable by the assay was 2 μg, a quantity that would be expected to generate 1.0 pmole angiotensin per minute from the natural plasma substrate. Chymotrypsin, trypsin, papain, and pseudorenin, were also effective in cleaving labeled peptides from the insoluble substrate, but leucine aminopeptidase did not appear to release soluble radioactivity. The assay, as described, is useful for the measurement of large numbers of renin samples because of the speed and ease with which it may be performed. It is not yet sufficiently sensitive nor specific to measure the low levels of renin found in plasma.     

Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F

The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P₁ and P₃′ residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a K_i value of 4 μM. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.     

Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in X-Lys-Gly sequences. We report here on the isolation of cDNA clones coding for the enzyme from a chick embryo lambda gt11 library. Several overlapping clones covering all the coding sequences of the 4-kilobase mRNA and virtually all the noncoding sequences were characterized. These clones encode a polypeptide of 710 amino acid residues and a signal peptide of 20 amino acids. The polypeptide has four potential attachment sites for asparagine-linked oligosaccharides and 9 cysteine residues, at least one of which is likely to be involved in the binding of the Fe2+ atom to a catalytic site. A surprising finding was that no significant homology was found between the primary structures of lysyl hydroxylase and prolyl 4-hydroxylase in spite of the marked similarities in kinetic properties between these two enzymes. A computer-assisted comparison indicated only an 18% identity between lysyl hydroxylase and the alpha-subunit of prolyl 4-hydroxylase and a 19% identity between lysyl hydroxylase and the beta-subunit of prolyl 4-hydroxylase. Visual inspection of the most homologous areas nevertheless indicated the presence of several regions of 20-40 amino acids in which the identity between lysyl hydroxylase and one of the prolyl 4-hydroxylase subunits exceeded 30% or similarity exceeded 40%. Southern blot analyses of chick genomic DNA indicated the presence of only one gene coding for lysyl hydroxylase.     

Staphylococcal delta toxin stimulates endogenous phospholipase A2 activity and prostaglandin synthesis in fibroblasts

Delta toxin, one of at least four toxins produced by pathogenic strains of the skin bacterium Staphylococcus aureus, is an amphipathic polypeptide possessing hemolytic and cytolytic activity. Delta toxin stimulates high levels of phospholipase A₂ activity in 3T3 mouse fibroblasts with concomitant synthesis and release of prostaglandins. Alpha toxin, another hemolytic toxin produced by strains of S. aureus, did not stimulate phospholipase A₂ or prostaglandin release in these cells. Analysis of the release of lactate dehydrogenase and β-galactosidase (cytoplasmic and lysosomal marker enzymes, respectively) from delta-toxin-treated cells indicated that cytolytic concentrations of the toxin damage the cell-surface membrane more extensively than lysosomal membranes. During a 30 min exposure, delta toxin stimulated 3T3 cells to hydrolyze up to 32% of the lipids biosynthetically labeled by incorporation of [³H]arachidonic acid. A relatively high percentage of the free arachidonic acid formed in delta-toxin-treated 3T3 cells was converted to prostaglandins (up to 41.3% and 8.3% converted to chromatographically identifiable prostaglandins E₂ and F_2α, respectively, in 30 min), with optimal conversion occurring at sublytic toxin concentrations. The degree of activation of phospholipase A₂ in 3T3 cells by a range of concentrations of delta toxin correlates with cytotoxicity assessed by failure to exclude trypan blue dye. Analysis of the calcium dependency of the toxin-activated phospholipase A₂ was consistent with a cell-surface, Ca²⁺-dependent enzyme. The phospholipase A₂ exhibits a degree of specificity for substrate lipids containing polyunsaturated fatty acid residues which can serve as precursors for prostaglandin formation. Enzymatic activity was not inhibited by diisopropylfluorophosphate (5 mM), N-ethylmaleimide (5 mM) or p-bromophenacylbromide (0.1 mM). Delta toxin did not activate detectable phospholipase A₂ in subcellular preparations containing plasma membrane.     

Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells.

Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)