Search

Monoclonal antibodies of both the IgG and the IgM type were purified by hydroxylapatite HPLC (HA-HPLC) under very mild conditions. The IgM type antibodies, which were isolated from ascites fluid and separated from other proteins also by means of size exclusion HPLC. It was shown that the most frequently observed disadvantage of HA-HPLC, that is the relative short life of the columns (P. Steffen (1989) GIT Fachz. Lab., Suppl. 3/89 (Chromatogr.), 50-90), is due to microbial contamination rather than lower mechanical stability. In order to monitor column performance, a test was developed based on the use of standard proteins under isocratic separation conditions. This allows a direct comparison between the respective performances of columns made from different materials, hydroxylapatite or fluoroapatite, from different sources and with different particle sizes. A problem which often occurs with HA-HPLC in the case of IgM antibody isolation, namely precipitation of the antibodies at low salt concentrations at the beginning of a chromatographic run, was avoided by adding sodium chloride to both separation buffers.

SummaryThe study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding.Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D’). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA.The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F. Weak inhibition is exerted on association of mAb 2, 21, and 25 to epitope D. Amidolytic activity is suppressed only by B-chain specific antibody 22.

Two murine monoclonal antibodies produced against human urokinase-type plasminogen activator were characterized with respect to their antigen-binding specificity and their effects on urokinase activity and urokinase receptor binding. One of the antibodies binds to the protease domain of urokinase (Kass = 2.1 X 10(7) M-1). Antibody binding inhibits catalysis of plasminogen activation. It does not, however, affect amidolytic activity of urokinase towards the chromogenic substrate D-Val-Leu-Arg-p-nitroanilide. The antibody thus appears to interfere with plasminogen binding without directly affecting catalytically active amino acid residues of the enzyme. The other antibody binds to the aminoterminal fragment of urokinase (Kass = 1.0 X 10(7) M-1) and prevents binding of the enzyme to high affinity receptors on human granulocytes. Binding of this antibody neither influences plasminogen activation nor the amidolytic activity of urokinase. Both antibodies are potentially useful for the further analysis and manipulation of urokinase function.

Leberextrakt hat in Gegenwart von L-Glutamat eine hemmende Wirkung auf die anaerobe Glykolyse der Ehrlich-Mäuseascites-Carcinomzelle. Die hemmende Wirkung des Leberextraktes wird auf die Wirkung von Glutamat-Pyruvat-Transaminase zurückgeführt. Das bei der Glykolyse intermediär entstehende Pyruvat wird durch die Transaminierungs-Reaktion abgefangen und somit der Dehydrierung von DPNH entzogen. Die sinkende Konzentration an DPN ⨁ hat eine Hemmung der Triosephosphatdehydrierung zur Folge. Die Glykolysehemmung durch GPT ist aufhebbar durch Sauerstoff sowie durch Zugabe von Pyruvat, von DPN ⨁, von α-Ketoglutarat und von L-Alanin.

Es wird ein Verfahren zur Darstellung hoch gereinigter Glutamat-Pyruvat-Transaminase beschrieben. Die anaerobe Glykolyse in Ehrlich-Mäuseascites-Carcinomzellen wird durch hoch gereinigte GPT ebenso gehemmt wie durch Leberextrakt, wenn die gleiche Konzentration an GPT-Einheiten eingesetzt wird.

Nucleolar ‘80-S’ and ‘40-S’ preribosomes (containining 45-S and 21-S pre-rRNA, respectively), as well as cytoplasmic ribosomes, were isolated from Friend erythroleukemia cells. The presence of structural ribosomal proteins in the isolated particles was studied by using antisera against individual rat liver small ribosomal subunit proteins. The analysis is based on the established crossreactivity between rat and mouse ribosomes [F. Noll and H. Bielka (1970) Mol. Gen. Genet. 106, 106–113]. The identification of the proteins was achieved by two independent immunological techniques: the passive haemagglutination test and the enzyme immunoassay of electrophoretically fractionated proteins, blotted on nitrocellulose. All 17 proteins tested are present in cytoplasmic ribosomes. A large number of proteins (S3a, S6, S7, S8, S11, S13, S14, S18, S20, S23/24 and S25) are present in the ‘80-S’ pre-ribosome. Only two proteins (S3 and S21) are added during the formation of the ‘40-S’ preribosome in the nucleolus. Four proteins (S2, S19, S26 and S29) are added at later, possibly extranucleolar, stages of ribosome formation. The results obtained provide evidence for the sequential addition of proteins during the formation of the small ribosomal subunit in Friend erythroleukemia cells.