The described method for the turbidimetric determination of serum transferrin concentration has the following features: the ionic strength of the medium is very low, the antiserum used is purified by the manufacturer, 25 μΐ antiserum per determination is sufficient, excess antibody or antigen has no influence.We studied the relation between transferrin concentration and total iron binding capacity for human normal and variant transferrins. Furthermore, our results are compared with the radial immunodiffusion technique. Immunologische turbidimetrische Methode zur Bestimmung von Transferrin im SerumZusammenfassung: Die f r die Transferrinbestimmung im Serum beschriebene turbidimetrische Methode hat folgende Charakteristika: Die lonenst rke des Mediums ist sehr gering. Das benutzte Antiserum ist vom Hersteller gereinigt, 25 /i Antiserum pro Bestimmung sind ausreichend, es gibt keinen Einflu von Antik rper-oder Antigen berschu .Wir untersuchten die Beziehung zwischen Transferrinkonzentration und Gesamt-Eisenbindungskapazit t f r normales Transferrin und -Varianten des Menschen. Die Ergebnisse der turbidimetrischen Methode wurden mit durch radiale Immundiffusion erhaltenen verglichen.

A series of hepatitis B virus open reading frame (ORF)preS-S variants, including mutants in which the relative order of the in-frame start codons (AUG1, AUG2 and AUG3) and nearby sequences had been altered, was expressed both in vivo (in HepG2 hepatoblastoma cells) and in vitro (by T7 promoter-driven transcription followed by translation in a rabbit reticulocyte lysate). The ratio of the synthesis of the large, middle (M) and major (S) proteins or their chimeric counterparts was analysed to study the translational regulation of ORF expression. As expected on the basis of the ribosome scanningmodel, the AUG sequence context was found to be a prominent factor in determining the different translational behaviour of the two preS-S-specific mRNAs of 2-4 kb (predominantly translated from AUG1) and 2.1 kb (which includes AUG2 and/or AUG3 and can be translated from either). Results from both experimental systems suggested that initiation at internal AUGs in the 2.4 kb RNA is possible. In experiments in vitro, preSS mutants bearing lesions in a region 5' to AUG2, which has been implicated in AUG2/AUG3 cis repression, showed no increase in the utilization of internal AUGs. In addition, the chimeric envelope polypeptides produced in transfected HepG2 cells in this study were informative with respect to preS-mediated endoplasmic retention: replacement of the preS2 N terminus with that from preS1 generated a chimeric M protein that was glycosylated within the putative preS 1 retention sequence ad was not secreted. Thus, the preS1 retention sequence most likely acts inside the lumen of endoplasmic reticulum and its function is insensitive to glycosylation. A similar element might be active at the N terminus of M protein.

To study the mechanism of L protein-mediated, intracellular (pre-Golgi) retention of hepatitis B virus (HBV) surface proteins, a collection of HBV preS-S open reading frame variants bearing wild-type or modified preS extensions was expressed in human cells. When the secretion phenotype of the corresponding proteins was analysed, all surface proteins with rearranged preS domains were found to be at least partially retained. This held true, in particular, for two variant proteins lacking preS1 amino acids 1 to 19 (ayw), the preS1 myristylated N terminus and a putative retention domain, and for another variant lacking the entire preS1 domain plus the N-terminal portion (amino acids 1 to 12) of the preS2 domain. All the retained variants underwent intracellular dimerization/oligomerization via disulphide bonds to a degree comparable to that observed in well exported natural proteins. Our results show that retention can take place in the absence of L Nterminal sequences and does not imply inhibition of covalent oligomerization.

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