An improved purification procedure for cyclosporine synthetase

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using 14C-labeled S-adenosyl-L-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-14C]AdoMet or [carboxyl-14C]AdoMet yielded the sequence H2N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu10654 and Pro10655 as the major sites of derivatization. [carboxyl-14C]AdoMet in addition labeled Tyr10650. Chymotryptic digestion generated the radiolabeled peptide H2N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu2128 and Pro2129, which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.

Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase

The multifunctional polypeptide cyclosporin synthetase (CySyn) remains one of the most complex nonribosomal peptide synthetase described. In this study we used a highly specific photoaffinity labeling procedure with the natural cofactor S-adenosyl-l-methionine (AdoMet), 14C-isotopically labeled at the Sδ methyl group to probe the concerted AdoMet-binding interaction of the N-methyltransferase (N-MTase) centers of CySyn. The binding stoichiometry for the enzyme–AdoMet complex was determined to be 1:7, which is in agreement with inferences made from analysis of the complementary DNA sequence of the simA gene encoding the CySyn polypeptide. The photolabeling of the AdoMet-binding sites displayed homotropic negative cooperativity, characterized by a curvilinear Scatchard plot with upward concavity. Although, the process of N-methyl transfer is not a critical event for peptide elongation, the destabilizing homotropic interactions between N-MTase centers that were observed may represent a mechanism whereby the enzyme preserves the proficiency of the substrate-channeling process of cyclosporin peptide assembly over a broad range of cofactor concentrations. Furthermore, we demonstrated the utility of the photolabeling procedure for tracking the enzyme during purification.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Nonribosomal peptide synthetases as technological platforms for the synthesis of highly modified peptied bioeffectors - cyclosporin synthetase as a complex example

Many microbial peptide secondary metabolites possess important medicinal properties, of which the immunosuppressant cyclosporin A is an example. The enormous structural and functional diversity of these low-molecular weight peptides is attributable to their mode of biosynthesis. Peptide secondary metabolites are assembled non-ribosomally by multi-functional enzymes, termed non-ribosomal peptide synthetases. These systems consist of a multi-modular arrangement of the functional domains responsible for the catalysis of the partial reactions of peptide assembly. The extensive homology shared among NRPS systems allows for the generalisation of the knowledge garnered from studies of systems of diverse origins. In this review we shall focus the contemporary knowledge of non-ribosomal peptide biosynthesis on the structure and function of the cyclosporin biosynthetic system, with some emphasis on the re-direction of the biosynthetic potential of this system by combinatorial approaches.

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus, were characterized using autoradiographical, molecular, and physiological techniques. Specific ¹²⁵I-rat ANP binding sites were present in the carotid and pulmonary arteries, the lateral aorta, the pre- and post-cava, and the jugular vein, and generally occurred in each layer of the blood vessel. The ¹²5I-rat ANP binding was partially displaced by the specific natriuretic peptide receptor C ligand, C-ANF, which indicates the presence of two types of natriuretic peptide receptors in the blood vessels. This was confirmed by a RT-PCR study, which demonstrated that guanylyl cyclase receptor (NPR-GC) and NPR-C mRNAs are expressed in arteries and veins. An in vitro guanylyl cyclase assay showed that frog ANP stimulated the production of cGMP in arterial membrane fractions. Physiological recordings from isolated segments of the carotid and pulmonary arteries and the lateral aorta, which had been pre-constricted with arginine vasotocin, showed that rat ANP, frog ANP and porcine CNP relaxed the vascular smooth muscle with relatively similar potency. Together, the data show that the central vasculature contains two types of natriuretic peptide receptors (NPR-C and NPR-GC) and that the vasculature is a target for ANP and CNP.

Functional analysis of natriuretic peptide receptors in the bladder of the toad, bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific ¹²⁵I-rat atrial natriuretic peptide (¹²⁵I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with ¹²⁵I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to ¹²⁵I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Toad atrial natriuretic peptide : cDNA cloning and functional analysis in isolated perfused kidneys

Complementary DNA (cDNA) encoding Bufo marinus (toad) preproatrial natriuretic peptide (preproANP) was isolated by reverse-transcription polymerase chain reaction. Sequence analysis of toad preproANP cDNA revealed an open reading frame of 150 amino acid residues, which shared 72% and 66% identity with Rana catesbeiana and Xenopus laevis preproANP, respectively. The deduced amino acid sequence of toad ANP that corresponded to ANP 1–24 of R. catesbeiana and Rana ridibunda was identical, but it differed by four residues from that of X. laevis. ANP mRNA transcripts were also shown to be expressed in the toad kidney. Subsequently, the effect of frog ANP (1–24) on renal function in toad was examined using a perfused kidney preparation. The arterial infusion of frog ANP caused a dose-dependent decrease in the arterial perfusion pressure that was associated with an increase in the glomerular filtration rate (GFR) and a renal natriuresis and diuresis. The renal natriuresis and diuresis resulted predominantly from an increased GFR rather than from direct tubular effects. This study demonstrates that ANP can regulate renal function, which suggests it may be involved in overall fluid volume regulation.

In situ localization associates biologically active plant natriuretic peptide immuno-analogues with conductive tissue and stomata

Plant natriuretic peptide immuno-analogues (irPNP) have previously been shown to affect a number of biological processes including stomatal guard cell movements, ion fluxes and osmoticum-dependent water transport. Tissue printing and immunofluorescent labelling techniques have been used here to study the tissue and cellular localization of irPNP in ivy (Hedera helix L.) and potato (Solanum tuberosum L.). Polyclonal antibodies active against human atrial natriuretic peptide (anti-hANP) and antibodies against irPNP from potato (anti-StPNP) were used for immunolabelling. Tissue prints revealed that immunoreactants are concentrated in vascular tissues of leaves, petioles and stems. Phloem-associated cells, xylem cells and parenchymatic xylem cells showed the strongest immunoreaction. Immunofluorescent microscopy with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG supported this finding and, furthermore, revealed strong labelling to stomatal guard cells and the adjacent apoplastic space as well. Biologically active immunoreactants were also detected in xylem exudates of a soft South African perennial forest sage (Plectranthus ciliatus E. Mey ex Benth.) thus strengthening the evidence for a systemic role of the protein. In summary, in situ cellular localization is consistent with physiological responses elicited by irPNPs reported previously and is indicative of a systemic role in plant homeostasis.

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.

Comparative aspects of natriuretic peptide physiology in non-mammalian vertebrates: a review

The natriuretic peptide system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. A natriuretic peptide system is present in each vertebrate class but there are varying degrees of complexity in the system. In agnathans and chondrichthyians, only one natriuretic peptide has been identified, while new data has revealed that multiple types of natriuretic peptides are present in bony fish. However, it seems in tetrapods that there has been a reduction in the number of natriuretic peptide genes, such that only three natriuretic peptides are present in mammals. The peptides act via a family of guanylyl cyclase receptors to generate the second messenger cGMP, which  mediates a range of physiological effects at key targets such as the gills, kidney and the cardiovascular system. This review summarises the current knowledge of the natriuretic peptide system in non-mammalian vertebrates and discusses the physiological actions of the peptides.

Identification of an α-peptide in Haliotis rubra with homology to the Lymnaea α-CDCP spawning peptide

It is now understood that a combination of molluscan reproductive peptides are commonly cleaved from a large preprohormone and influence different aspects of spawning behavior. One type of reproductive peptide, known in Lymnaea stagnalis as [alpha]-CDCP, and in Aplysia californica as [alpha]-BCP, acts in egg laying via temperature-dependent autoinhibition or autoexcitation of neuronal cells. In our study, the expression of [alpha]-CDCP-like peptide in the blacklip abalone, Haliotis rubra, was identified by Western blots and immunocytochemistry, using an antiserum developed against [alpha]-CDCP. Western blots of total protein isolated from the central nervous system, cerebral and pleuropedal ganglia, as well as gonad and heptopancreas tissues of sexually mature adults, identified a protein of approximately 100 kDa as well as a range of smaller reactive peptides. This finding suggests that a reproductive [alpha]-peptide is probably synthesized from a single larger precursor protein. The larger peptides were also identified in Western blots of several abalone tissues, lmmunocytochemistry using the same antiserum showed the presence of immunoreactive axons in all the tissues studied, indicating synthesis or transport of products. The function of the abalone [alpha]-CDCP-like peptide is yet to be determined.

Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, Anguilla japonica

Natriuretic peptides are linked to osmoregulation, cardiovascular and volume regulation in fishes. The peptides bind to two guanylyl-cyclase-linked receptors, natriuretic peptide receptor-A (NPR-A) and NPR-B, to elicit their effects. Atrial natriuretic peptide (ANP) binds principally to NPR-A, whereas C-type natriuretic peptide (CNP) binds to NPR-B. The teleost kidney has an important role in the maintenance of fluid and electrolyte balance; therefore, the location of NPR-A and NPR-B in the kidney could provide insights into the functions of natriuretic peptides. This study used homologous, affinity purified, polyclonal antibodies to NPR-A and NPR-B to determine their location in the kidney of the Japanese eel, Anguilla japonica. Kidneys from freshwater and seawater acclimated animals were fixed overnight in 4% paraformaldehyde before being paraffin-embedded and immunostained. NPR-A immunoreactivity was found on the apical membrane of proximal tubule 1 and the vascular endothelium including the glomerular capillaries. In contrast, NPR-B immunoreactivity was located on the smooth muscle of blood vessels including the glomerular afferent and efferent arterioles, and on smooth muscle tissue surrounding the collecting ducts. No difference in the distribution of NPR-A and NPR-B was observed between freshwater and seawater kidneys. Immunoreactivity was not observed in any tissue in which the antibodies had been preabsorbed. In addition, there was no difference in NPR-A and NPR-B mRNA expression between freshwater-acclimated and seawater-acclimated eels. These results suggest that, although utilizing the same second messenger system, ANP and CNP act on different targets within the kidney and presumably elicit different effects.

The response of the natriuretic peptide system to water deprivation in the desert rodent, Notomys alexis

Natriuretic peptides (NPs) are regulatory molecules that cause cGMP-mediated diuresis and natriuresis in mammals. Accordingly, it is interesting to consider their role in desert-adapted animals in which water is often limited. This study investigated the response of the natriuretic peptide (NP) system to varying periods of water deprivation (WD) in the Australian desert rodent species, Notomys alexis. It was hypothesised that the expression of the NP system will be down-regulated in water-deprived N. alexis compared to water-replete animals. The plasma levels of ANP were significantly reduced after 3 days of WD, but were unaffected by 7, 14 and 28 days of WD. Water deprivation for 3, 7, 14 days had a variable effect on the mRNA expression of ANP, CNP, NPR-A, NPR-B, and NPR-C, and a uniform down-regulation was not observed. However, after 28 days of WD, mRNA expression was similar to water-replete animals, except for NPR-A. Surprisingly, 7 and 14 days of WD caused an up-regulation in the ability of ANP to stimulate cGMP; this also occurred at 14 days for CNP. Taken together, the mRNA expression and peptide mediated guanylyl cyclase activity data after WD were in the opposite direction to what was predicted. Interestingly, after 28 days of WD, most parameters were similar to those of water-replete animals, which indicates that a down-regulation of the NP system is not part of the physiological response to an absence of free water in N. alexis.