Lambro A. Johnson

Adenine phosphoribosyltransferase: a simple spectrophotometric assay and the incidence of mutation in the normal population.

The significance of partial deficiency of erythrocyte adenine phosphoribosyltransferase (APRT), reported in a number of subjects with gout, has been investigated by studying its incidence in 700 normal blood donors. Three clearly deficient subjects were found, an incidence not significantly different from that in patients with abnormalities of urate metabolism. A new assay method for APRT is described in which an erythrocyte lysate is incubated with adenine and phosphoribosylpyrophosphate (PRPP) for a given time; both hemoglobin and adenine nucleotide (AMP) are then precipitated with lanthanum phosphate; the change in absorbance of adenine at 260 nm reflects the extent of its conversion to AMP by APRT.

Textilinin-1, an alternative anti-bleeding agent to aprotinin: Importance of plasmin inhibition in controlling blood loss.

Aprotinin has been used widely in surgery as an anti‐bleeding agent but is associated with a number of side effects. We report that textilinin‐1, a serine protease inhibitor from Pseudonaja textilis venom with sequence relatedness to aprotinin, is a potent but reversible plasmin inhibitor and has a narrower range of protease inhibition compared to aprotinin. Like aprotinin, textilinin‐1 at 5 μmol/l gave almost complete inhibition of tissue plasminogen activator‐induced fibrinolysis of whole blood clots. The activated partial thromboplastin time for plasma was markedly increased by aprotinin but unaffected by textilinin‐1. In a mouse tail‐vein bleeding model, intravenous textilinin‐1 and aprotinin caused similar decreases in blood loss but time to haemostasis in the textilinin‐treated animals was significantly shorter than in aprotinin‐treated mice. Based on these data, textilinin‐1 merits further investigation as a therapeutic alternative to aprotinin.

Comparison of Textilinin-1 with Aprotinin as Serine Protease Inhibitors and as Antifibrinolytic Agents

Textilinin-1 (Q8008) was isolated from the venom of the Pseudonaja textilis and has a 47% sequence identity to the antihaemorrhagic therapeutic agent aprotinin. When equimolar concentrations of enzyme and aprotinin were pre-incubated, plasmin was inhibited 100%, plasma kallikrein 58%, and tissue kallikrein 99%. Under the same conditions, textilinin-1 inhibited plasmin 98%, plasma kallikrein 16% and tissue kallikrein 17%. Whole blood clot lysis was inhibited strongly by both aprotinin and textilinin-1, as shown by thrombelastography. At 2 µM inhibitor lysis initiated by t-PA was greater than 99% inhibited by aprotinin (LY60 = 0.4 ± 0.1) whereas textilinin-1, inhibited lysis by 91% (LY60 = 8.9 ± 0.7). The same trend was found with the lysis of euglobulin fractions. From these data textilinin-1 appears to be a more specific plasmin inhibitor than aprotinin but aprotinin inhibits clot lysis to a greater extent.

Regulation of purine de novo synthesis in cultured human fibroblasts: The role of P-Ribose-PP

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.

The structure of human microplasmin in complex with textilinin-1, an aprotinin-like inhibitor from the Australian brown snake

Textilinin-1 is a Kunitz-type serine protease inhibitor from Australian brown snake venom. Its ability to potently and specifically inhibit human plasmin (Ki = 0.44 nM) makes it a potential therapeutic drug as a systemic anti-bleeding agent. The crystal structures of the human microplasmin-textilinin-1 and the trypsin-textilinin-1 complexes have been determined to 2.78 Å and 1.64 Å resolution respectively, and show that textilinin-1 binds to trypsin in a canonical mode but to microplasmin in an atypical mode with the catalytic histidine of microplasmin rotated out of the active site. The space vacated by the histidine side-chain in this complex is partially occupied by a water molecule. In the structure of microplasminogen the χ1 dihedral angle of the side-chain of the catalytic histidine is rotated by 67° from its “active” position in the catalytic triad, as exemplified by its location when microplasmin is bound to streptokinase. However, when textilinin-1 binds to microplasmin the χ1 dihedral angle of this amino acid residue changes by −157° (i.e. in the opposite rotation direction compared to microplasminogen). The unusual mode of interaction between textilinin-1 and plasmin explains textilinin-1′s selectivity for human plasmin over plasma kallikrein. This difference can be exploited in future drug design efforts.

Hypoxanthine-guanine phosphoribosyltransferase: A simple spectrophotometric assay

A simple spectrophotometric assay is described based on the conversion of hypoxanthine to inosine monophosphate and precipitation of both the reaction product and protein with lanthanum phosphate. The extent of conversion is determined by the fall in absorbance of hypoxanthine at 249 nm. The assay is suitable for screening red cell lysates for hypoxanthine-guanine phosphoribosyltransferase deficiency.

Incidence of APRT Deficiency

A partial eficiency of adenine phosphoribosyltransferase (APRT) has been reported in a number of subjects with gout, as well as in asymptomatic individuals (1–4). The association with gout has been attributed to the chance association of two common conditions and no mechanism has been demonstrated to relate APRT deficiency to a disorder of purine metabolism. The incidence of APRT deficiency in 700 normal subjects was assessed to test this hypothesis. A spectrophotometry assay was developed which was ideally suited for large scale surveys. The assay was based on the change of UV-absorbance which resulted on the conversion of Ad to its nucleotide and was dependent upon the simultaneous and quantitative removal of hemoglobin and AMP from the incubation solution. No significant difference was found between the values obtained by this method and the standard radiochemical method (5).

Inactivation of Hypoxanthine Guanine Phosphoribosyltransferase by Guanosine Dialdehyde : An Active Site Directed Inhibitor

Congenital deficiencies of the enzyme HGPRT are invariably associated with metabolic overproduction of purines, which manifest clinically as hyperuricaemia and gout (1). In cases of severe enzyme deficiency (Lesen Nyhan syndrome), these manifestations are accompanied by a bizarre neurological disorder (2). Production of the enzyme deficiency in laboratory animals would be a considerable experimental advantage in studying these disorders. This might be achieved by the use of active-site directed irreversible inhibitors of the enzyme (3). Although a wide range of substrate analogues have been synthesised, most have exhibited poor binding compared to the substrates. A more successful approach has been the modification of the enzyme reaction product GMP. GMP-dialdehyde, the periodate oxidation product of GMP, has been shown to be a specific irreversible inhibitor of HGPRT in cell extracts but was unable to penetrate the intact cell membrane (4, 5). The experiments reported here show that guanosine dialdehyde (GDA), the periodate oxidation product of guanosine, is also a relatively specific but less potent inhibitor of HGPRT and appears to be able to act intracellularly.

De Novo Purine Synthesis in Cultured Human Fibroblasts

In mammalian cells, purine nucleotides may be formed via (a) de novo synthesis and (b) salvage pathways. Both these pathways utilise PRPP and the concentration of this substrate is thought to be important in regulating the rate of purine synthesis (1, 2). Individuals with a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT), have increased production of uric acid (3, 4). Lymphocyte and fibroblast cultures of HPRT-deficient (HPRT-) cells have been reported as having accelerated rates of purine de novo synthesis associated with elevated concentrations of PRPP (5, 6).

Next-generation rapid serum tube technology using prothrombin activator coagulant: fast, high-quality serum from normal samples

Abstract Background Incomplete blood clotting or latent clotting in serum is a common laboratory problem, especially for patients on anticoagulant therapy or when serum tubes are centrifuged before clotting is completed. We describe a novel approach to producing high-quality serum using snake venom prothrombin activator complex (OsPA) as an additive in blood collection tubes for non-anticoagulated (normal) individuals. Methods Plasma clotting assays were performed using a Hyland-Clotek instrument. Blood clotting was visually observed, and thromboelastography was also performed to determine the important parameters of coagulation. Thrombin generation was assayed using the chromogenic substrate S-2238, and biochemical analytes in the serum were determined on chemistry and immunoassay analysers. Fibrinogen was determined by either ELISA or Clauss fibrinogen assay. Results We initially showed that OsPA had strong coagulation activity in clotting not only recalcified citrated plasma and recalcified citrated whole blood, but also fresh whole blood in a clinical setting. The use of TEG clearly showed improved speed of clotting and generation of a firmer clot. We also showed that the use of OsPA to produce serum did not interfere with the determination of commonly measured biochemical analytes. The underlying clotting mechanism involves a burst of thrombin production at the initial stages of the clotting process upon contact with prothrombin in blood. Conclusions These results demonstrate rapid generation of high-quality serum, contributing to faster turnaround times with standardised quality samples, for accurate analyte determinations in normal individuals.