Kynureninase E.C.3.7.1.3. is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver ...kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The
K
m values of the rat enzyme, for
l-kynurenine and
dl-3-hydroxykynurenine, were 440±20 μM and 32±5 μM and of the human enzyme 440±20 μM and 49±6 μM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate β-lyase activity.
The kinetic behavior of a typical Hill reaction catalyzed by thylakoids and using the oxidized form of 2,6‐dichloroindophenol (DCPIPox) as the artificial electron acceptor, is considered. Here, the ...light absorption process and the reduction of DCPIPox are autocatalytically coupled, leading to the occurrence of multiple steady states with respect to either the acceptor concentration or the incident light intensity. Experimental evidence is presented for both cases and the emergence of autocatalysis is discussed. The effect of the spatial arrangment on the global behavior of the system described is emphasized.
The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of
l-kynurenine to kynurenic acid. A combination of polymerase chain reaction techniques and hybridization screening was used to ...isolate a cDNA clone encompassing the entire coding region of KAT from rat kidney. Identification of the cDNA as coding for KAT was based both on the comparison of amino acid sequences obtained from purified rat KAT and on the expression of KAT activity in COS-1 cells transfected with the cDNA. RNA blot analysis indicated that KAT mRNA is widely expressed in rat tissues. Cultured cells transfected with the cDNA for KAT also showed glutamine transaminase K activity. Based mainly on sequence data, these results demonstrate that rat kidney KAT is identical with glutamine transaminase K.
A new mutein of interleukin-6, called δ22-Il-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys
23 and ...Cys
29) with serine residues, was produced in
Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (p
I 6.10) and a more basic component (p
I 6.70), the native form having a p
I of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the p
I 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher p
I form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the p
I 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the p
I 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.
Pro‐urokinase is a natural plasminogen activator that displays a clot‐lysis activity through a fibrin‐dependent mechanism. It seems to be a promising agent for the treatment of coronary thrombosis. ...Like tissue‐type plasminogen activator and two‐chain urokinase‐type plasminogen activator, pro‐urokinase has a very short half‐life in circulation.
It has been described that conjugation of serum albumin with pro‐urokinase in plasma may occur that could protect this protein from degradation.
In this study we describe the insertion of an extra cysteine residue in the N‐terminal end of des‐(C11–K135)‐pro‐urokinase (Δ125‐proUK), a pro‐urokinase deletion mutant lacking amino acids 11–135. We have expressed and purified the new mutein H5K, S9C, N10Tdes‐(C11‐K135)‐pro‐urokinase (Cys‐Δ125‐pro‐urokinase) and chemically conjugated it with serum albumin via the extra cysteine of Cys‐Δ125‐pro‐urokinase.
The purified conjugate obtained has a lower specific amidolytic activity (72000 U/mg) than unconjugated Cys‐Δ125‐pro‐urikinase (240000 U/mg) due to its higher molecular mass and has a similar fibrinolytic activity in a clot lysis test to that of Δ125‐pro‐urokinase.
We established an ELISA to measure the concentration of the conjugate in plasma and to follow the pharmacokinetics of the conjugate in monkeys after bolus injection.
The conjugate displays significant lysis of human plasma clots in vivo and a dramatic increase of the half‐life in the circulation, with respect to pro‐urokinase and Δ125‐pro‐urokinase. Therefore, preliminary biological characterisation of this conjugate indicates that it could be a good candidate to inject as a bolus, compared with the infusion regimen needed with pro‐urokinase.
The coupling of isoelectric focusing on immobilized pH gradients (IPG) with electrospray-mass spectrometry (ES-MS) was applied to the characterization of proteins according to two different and ...important properties, such as net surface charge and molecular mass. From a technical point of view, these methods are complementary, since ES-MS requires ion-free samples as usually supplied by isoelectric focusing on IPGs. This report describes the experiments carried out on model proteins to demonstrate the feasibility of the sequential application of these two techniques for the characterization of proteins. A minimum of 5 micrograms protein was needed for good signal by mass spectrometry. The following proteins were studied: myoglobin, truncated interleukin-6-mutein, recombinant cytochrome c551 and insulin-like growth factor I. Extraction from the IPG matrix was carried out in 70% acetonitrile/30% water/0.05% trifluoroacetic acid either by passive diffusion or by centrifugation through a 0.22 micron Amicon membrane, with protein recoveries of 80-85%.
Kynurenic acid (KA) is an endogenous glutamate receptor antagonist at the level of the different ionotropic glutamate receptors. One of the enzymes responsible for the production of KA, kynurenine ...aminotransferase I (KATI), also catalyses the reversible transamination of glutamine to oxoglutaramic acid (GTK, EC 2.6.1.15). The enzyme exists in a cytosolic and in a mitochondrial form because of the presence of two different KATI mRNAs coding for a protein respectively with and without leader sequence targeting the protein into mitochondria. We have cloned from a phage library of rat kidney cDNA four new KATI cDNAs containing different 5' untranslated regions (UTRs). One of the transcripts (+14KATI cDNA) contains an alternative site of initiation of translation. The tissue distribution of the different transcripts was studied by RT-PCR. The study demonstrated that several KATI mRNAs are constitutively expressed in ubiquitous manner, while +14KATI mRNA is present only in kidney. The translational efficiency of the different transcripts was studied in vitro and enzymatic activities were measured in transiently transfected Cos-1 cells. Each KATI mRNA exhibits a different in vitro translational efficiency, which corresponds to different levels of KAT enzymatic activity in transfected cells. Both findings correlate with the predicted accessibility of the ribosomal binding sites of the different mRNAs. The structure of the rat KATI/GTK gene was also studied. The expression of several KATI mRNAs with different 5'UTRs represents an interesting example of transcriptional/translational control on the expression of pyridoxal phosphate (PLP)-dependent aminotransferases.
A mutant species of the 185‐residue chain of human interleukin‐6 lacking 22‐residues at its N‐terminus and with a Cys→Ser substitution at positions 45 and 51 was produced in Escherichia coli. The ...163‐residue protein des‐(A1–S22)‐C45S, CS1Sinterleukin‐6, containing a single disulfide bridge, formed inclusion bodies. Mutant interleukin‐6 was solubilized in 6 M guanidine hydrochloride, subjected to oxidative refolding and purified to homogeneity by ammonium sulfate precipitation and hydrophobic chromatography. The purity of the mutant species was established by electrophoresis, isoelectrofocusing and reverse‐phase HPLC and its structural identity was checked by N‐terminal sequencing of both the intact protein and several of its proteolytic fragments. Electrospray mass spectrometry analysis of mutant interleukin‐6 gave a molecular mass of 18 695 ± 2 Da in excellent agreement with the calculated value. Circular dichroic, fluorescence emission and second‐derivative ultraviolet absorption spectra indicated that mutant interleukin‐6 maintains the overall secondary and tertiary structure, as well as stability characteristics, of the recombinant wild‐type human interleukin‐6. The urea‐induced unfolding of mutant interleukin‐6, monitored by circular dichroic measurements in the far‐ultraviolet region, occurs as a highly cooperative process with a midpoint of denaturation at 5.5 M urea. The data of the reversible unfolding of mutant interleukin‐6 mediated by urea were used to calculate a value of 20.9 ± 0.4 kJ · mol−1 for the thermodynamic stability of the protein at 25°C in the absence of denaturant. The biological activity of mutant interleukin‐6 was evaluated in vitro by the hybridoma proliferation assay, and in vivo by measuring thrombopoiesis in monkeys. Dose/response effects of the mutant were comparable or even higher than those of the wild‐type protein. Overall the results of this study show that mutant interleukin‐6 is a biologically active cytokine, which could find practical use as a therapeutic agent.
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