Naturally-occurring spinosyn A and its derivatives function as argininosuccinate synthase activator and tumor inhibitor.

Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.

Citrullinaemia type I: a common mutation in the Pacific Island population.

AIM: The aim of this study was to develop and apply a mutation screening protocol for the ASS1 gene in order to confirm the diagnosis of citrullinaemia type I in neonates with elevated citrulline on expanded newborn screening (E-NBS). METHODS: Three patients with an elevated citrulline level were identified via routine E-NBS between January and October 2008. Analysis of the ASS1 gene using a polymerase chain reaction and sequencing-based method was successfully applied to all three patients, together with a rapid mutation-specific detection method. Their clinical progress was followed for 16-22 months. RESULTS: All three patients were homozygous for a previously reported missense mutation, c.787G>A (p.Val263Met), associated with a mild or asymptomatic clinical course. CONCLUSIONS: As a consequence of E-NBS, an increasing number of neonates with elevated citrulline of uncertain clinical significance are being identified. Rapid sequence analysis of the ASS1 gene can be used to confirm citrullinaemia type I and, increasingly, to infer phenotypic severity. Homozygosity for the same mutation was found in all three patients despite non-consanguinity and variable Pacific Island origin. These data suggest that this mutation may be relatively prevalent in these ethnic groups and imply a possible founder effect.

S-SAD, Se-SAD and S/Se-SIRAS using Cu Kalpha radiation: why wait for synchrotron time?

The structure of Escherichia coli argininosuccinate synthetase (EAS) has been determined using S-SAD, Se-SAD and S/Se-SIRAS data measured with Cu Kalpha radiation. EAS contains 16 methionines and three cysteines in 455 amino acids. At a wavelength of 1.54 A (Cu Kalpha), the native (S-Met) and derivative (Se-Met) proteins yield anomalous signals of approximately 0.86 and 1.6%, respectively. Highly redundant data were measured to 2.0 A from native and derivative EAS crystals. All three structure determinations were carried out in a highly automated manner using SnB and SOLVE/RESOLVE. Despite the minute Bijvoet differences at 1.54 A, the signal was sufficient to determine the heavy-atom substructure and produce high-quality electron-density maps in all three cases. These maps were readily interpretable by the RESOLVE automated building algorithm, which modeled greater than 75% of all three structures. The success of these methods has profound implications for crystallographers experiencing difficulty with heavy-atom incorporation or with limited access to a synchrotron source.

Expression, purification, crystallization and preliminary X-ray analysis of Escherichia coli argininosuccinate synthetase.

A recombinant form of Escherichia coli argininosuccinate synthetase with a C-terminal polyhistidine affinity tag has been expressed, purified and subsequently crystallized using the hanging-drop vapour-diffusion technique. The crystals grow as large rectangular chunks with unit-cell dimensions a = 79.70, b = 105.84, c = 127.33 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group I222 and diffract to a minimum d-spacing of 1.6 A at station X8C of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of density calculations, one monomer of this homotetrameric protein is predicted per asymmetric unit (Matthews coefficient V(m) = 2.69 A(3) Da(-1)).

Renal argininosuccinate synthetase: purification, immunohistochemical localization, and elastin-binding property.

Several proteins interact with elastin, an essential extracellular matrix element. We describe here an additional elastin-binding protein from the porcine kidney. This protein was extractable with an isotonic buffer, and was purified by a rapid and simple procedure employing its characteristic properties: high affinity for red A-agarose and spontaneous formation of cryoprecipitate. Immunohistochemical studies using antibody against the purified protein demonstrated its localization in the cytoplasm of proximal tubules of the normal rat kidney. On Western blotting using the antibody, an immunoreactive protein was detected in the liver as well as in the kidney. On the basis of the partial amino acid sequences of the purified protein, it was identified as argininosuccinate synthetase. Some biochemical properties of the protein were different from the data reporting a hepatic form of argininosuccinate synthetase, a key enzyme in the urea cycle in the liver. These results suggest that the elastin-binding property of a renal form of the enzyme may be related to a tissue-specific function in the kidney.

Argininosuccinate synthetase of bovine liver: chemical and physical properties.

Homogeneous crystalline argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] prepared from bovine liver according to Rochovansky et al. [Rochovansky, O., Kodowaki, H. & Ratner, S. (1977)J. Biol. Chem. 252, 5287-5294] has been characterized with respect to amino acid composition and other chemical and physical properties. The total residue molecular weights derived from the amino acid analysis are in agreement with values previously obtained by physical means for the catalytically active tetramer, 185,000, and the monomer, 46,500. The enzyme is focused sharply at pH 7.6 as a single protein. Additional properties reported include 2.74 X 10(5) M-1 cm-1 for the molar absorption coefficient, based on the absolute value for protein, and 0.747 ml/g for the chemically based partial specific volume.

[Physico-chemical properties of guinea pig liver argininosuccinate synthetase (author's transl)]. / Propiedades físico-químicas de la arginosuccinato-sintetasa de hígado de cobaya.

The behaviour of guinea pig liver argininosuccinate synthetase with respect to pH, temperature and kinetic parameters of substrates and inhibitors has been investigated. The enzyme shows the maximum activity at pH 8 and maximum stability (15 min at 30 degrees C) at pH in the range of 6.5 and 8. With respect to temperature it remains stable (15 min at pH 8) up to 40 degrees C. The results found for the Km values of the enzyme substrates L-citrulline, L-aspartate and ATP are 0.038 mM, 0.045 mM and 0.090 mM respectively. L-ornithine, diaminopimelic acid, pyrophosphate and ATP acted as inhibitors of the enzyme (competitive or not, according to the case). These kinetic studies of substrates and inhibitors were carried out with a partially purified fraction of the enzyme which had been purified 7.3 fold, which practically suppressed the ATP-ase activity, present in crude extracts.

Isolation and characterization of argininosuccinate synthetase from human liver.

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.

Yeast argininosuccinate synthetase. Purification; structural and kinetic properties.

Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228,000 as determined by gel sieving. It is composed of identical subunits of Mr 49,000 as shown by gel electrophoresis. The quaternary structure as determined by cross-linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric. The saturation functions by citrulline and aspartate are hyperbolic; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and aspartate; it is increased by argininosuccinate and arginine. Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead-end inhibition studies comply with a rapid-equilibrium random model, except for the interconversion of the central quaternary complexes; the different kinetic constants have been established on the basis. Yeast argininosuccinate synthetase has a double metabolic function: anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well-adjusted activity for both roles and to an economic and efficient utilization of ATP.