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Plasminogen-activator inhibitor type 1 (PAI-l), the primary physiological inhibitor of tissue-type plasminogen activator, is an unusual member of the serine protease inhibitor (serpin) superfamily in that it spontaneously converts to a latent form lacking activity. This latent form can be reactivated by denaturation and refolding, but the activation is usually incomplete and often leads to aggregation of the protein. In this study we have developed a high-level expression system that leads to the accumulation of PAI-1 at 30-50% total microbial protein. We have developed a single-step purification protocol which can be completed in a few hours, yielding approximately 20 mg purified recombinant PAI-Mitre culture. The purified PAI-1 was 80-100% active and was stable upon incubation at 37°C with a half-life of approximately 48 h. At 20°C, PAI-1 activity was stable for a week and at 4°C it retained its activity completely for up to two months. Freezing caused significant loss of activity. The stability of PAI-1 activity was found to be dependent on pH and ionic strength, being most stable at pH 5.6 and at an ionic strength of 1 M salt. We show that by a combination of highlevel expression and rapid purification under optimum conditions, it is possible to produce active and stable PAI-1 in high yield.Activation of plasminogen to form the serine protease plasmin is central to the processes of fibrinolysis [l], cell migration, angiogenesis and tumour metastasis [2]. Activation of plasminogen is accomplished by either tissue-type or urokinase-type plasminogen activator. Regulation of the system occurs at several levels, including assembly of the components and stimulation of activation on fibrin [l] or at cell surfaces [3, 41. The inhibition of the active enzymes is an important element in the control of the plasminogedplasmin system. Plasminogen-activator inhibitor type 1 (PAI-1) is the main physiological inhibitor of both tissue-type plasminogen activator and urokinase-type plasminogen activator, with association rate constants of the order of 0.1 pM s- ' [5]. PAI-1 is present in a wide variety of tissues and cultured cells, where regulation of its expression by several stimuli has been observed [6]. It is present in the circulation both in cell-free plasma and in platelets, these two pools being clearly distinct [7]. The plasma concentration of PAI-1 is normally low, approximately 20 ng/ml, but higher levels are observed in a range of diseases, suggesting an association with thrombotic disorders [S]. The platelet pool of PAI-1 is released during aggregation, so that it protects the newly formed thrombus against premature dissolution. Platelet PAI-1 contributes to the resistance PAI-1 was identified as a member of the superfamily of serine protease inhibitors known as serpins, on the basis of its sequence [16-181. The serpins are the major class of inhibitors regulating the fibrinolytic, coagulation and complement cascades. They act as pseudosubstrates and form an SDS-stable covalent 1 : 1 complex with their target...

Approximately 500 vitamin B6 auxotrophs were isolated from 18 independent cultures of Escheric/iia coli strain CR63. None grew in minimal medium supplemented with 2'-hydroxypyridoxine. Eighteen auxotrophs which had arisen independently were further characterized. All of them were defective in vitamin B6 synthesis rather than in an aminotransferase involved in vitamin B6 utilization. Two different phenotypes were recognized : ' Oxidase ' mutants which grew only when supplied with pyridoxal or pyridoxal 5'-phosphate and ' Pre Pn ' mutants which would also grow with pyridoxine or pyridoxine phosphate. ' Oxidase' mutants were confined to a single linkage group, but data from interrupted mating experiments established that 'Pre Pn' mutants fall into two linkage groups which are possibly identical to pdxA and pdxB. All mutations in the pdxA region were allelic rather than located in two closely linked genes.

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Glucosamine acted as a gratuitous catabolite repressor of maltose utilization in Saccharomyces cerevisiae. This repression was relieved in a linear manner as the maltose concentration was increased. Three mutants were isolated in which maltose utilization was no longer repressed by glucosamine. One of these mutants may be generally deficient in catabolite repression since it was not defective in glucosamine transport but was insensitive to glucosamine repression on all catabolite repressible carbon sources tested. Each mutant possessed a maltose uptake system which was notably less sensitive to glucose repression than that of the wild-type strain. These results are discussed in terms of a proposed model for regulation of maltose utilization in S. cerevisiae.