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Two-dimensional electrophoresis (2-DE) of cerebrospinal fluid (CSF) samples--from 347 patients with various psychiatric and neurological disorders--and subsequent silver staining revealed two additional polypeptides (Mr 40,000) in 49% of 111 schizophrenics, 46% of 43 schizoaffective patients, 36% of 41 patients with affective disorders, 43% of 28 patients with multiple sclerosis, but not in 25 patients without neurological symptomatology, nor in 9 patients with Lues, and in only 2 of 25 patients with AIDS. The two polypeptides, as detected by 2-DE, eluted after size exclusion chromatography in fractions containing proteins with Mr greater than 200,000. After 2-DE of CSF samples, enriched by gel chromatography, the polypeptides were immobilized by blotting onto glass-fiber membranes and subjected to N-terminal sequencing. Polypeptide A was identified as beta-chain remnant (beta 2), derived from plasmin cleavage of fibrin(ogen). After size exclusion chromatography, 2-DE, and Western blotting, polypeptide A and B, as well as several other spots, reacted with fibrinogen antibodies, suggesting that the polypeptides are subunits of a fibrin degradation complex.

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4x6-meric hemocyanin of the tarantula Eurypelma californicum was dissociated into subunits; one type of subunit was removed by immunoaffinity chromatography and replaced by its apo-or met-form. The mixture was reassembled and the reconstituted 4x6-mers were isolated. This was performed for subunits a, be, d, e, /and g, respectively. It was verified by crossed immunoelectrophoresis that each type of subunit including the modified one, was incorporated in the reassembled 4x6-mers. Oxygen binding curves of the purified reconstituted 4x6-mers were recorded at different pH values (pH 7.0-9.0; 20 °C). Halfsaturation pressures (p so ) and the cooperativities were calculated using unmodified, reassembled 4x6-mers as reference. In all cases, incorporation of a met-subunit increased oxygen affinity. In contrast, incorporation of an apo-subunit either slightly decreased oxygen affinity (be, /and d) or had no detected influence (others). The Bohr effect remained more or less unchanged in every case. Cooperativity was generally decreased. The metmodification of subunit d had the strongest effect. No significant differences could be observed between the respective met-and apomodification, except for experiments with subunit d. Generally, the value of hm^ exceeded h so by a factor of 1.3 to 1.5. pH-sensitivity of cooperativity was distinctly influenced depending on the modified subunit. The strongest effect was observed for subunit be. Our results demonstrate, for the first time, that each subunit of tarantula hemocyanin is involved in the allosteric processes. Apparently, they uniformly contribute to oxygen affinity and Bohr effect, but distinctly to cooperativity and pH sensitivity of the latter. Allosterische Sauerstoffbindungseigenschaften von reassoziiertem Vogelspinnen-Hämocyanin (Eurypelma californicum) mit eingebauten Apo-bzw. Met-UntereinheitentypenZusammenfassung: 4x6-meres Hämocyanin der Vogelspinne Eurypelma californicum wurde in Untereinheiten dissoziiert; ein Untereinheitentyp wurde mittels Affinitätschromatographie entfernt und durch die entsprechende Apobzw. Met-Form ersetzt. Das Gemisch wurde reassoziiert und rekonstituierte 4x6-mere isoliert. Der Vorgang wurde für die einzelnen Untereinheiten a, bc, d, e, f und g durchgeführt.Mittels zweidimensionaler Immunelektrophorese konnte nachgewiesen werden, daß jeder Untereinheitentyp, d.h. auch die modifizierten, in reassoziierte 4x6-mere eingebaut waren. Bei verschiedenen pH-Werten wurden Sauerstoffbindungskurven aufgenommen (pH 7.0-9.0; 20 °C). Halbsättigungsdrücke (p so ) und Werte für die Kooperativitäten wurden berechnet, wobei unmodifizierte reassoziierte 4x6-mere als Referenz dienten.