Delineation of a linear epitope by multiple peptide synthesis and phage display.

Two different approaches, the phage display technique and the Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by Fab 57P, specific for tobacco mosaic virus protein (TMVP), and define the preferred chemical composition of a functional epitope. Kinetic measurements of the interaction between peptide variants and the antibody fragment were used to further refine the molecular basis of binding activity. Our results show that the functional epitope of Fab 57P requires precise physico-chemical properties at a limited number of positions, and that residues flanking these key residues can influence binding affinity. The phage display and Spot synthesis methods allowed the straightforward localization of the binding region and the identification of residues that are essential for recognition. However, these methods yielded slightly different views of accessory factors that are able to influence antibody binding. The influence on binding activity of these factors can only be assessed through quantitative affinity measurements.

[Electrophoretic and enzymatic analysis of glycosaminoglycans in the blood serum of patients with hyperthyroidism]. / Analiza elektroforetyczna i enzymatyczna glikozoaminoglikanów w surowicy osób z nadczynnoscia tarczycy.

An effect of hyperthyroidism on the composition and levels of glycosaminoglycans in the blood serum was studied. Glycosaminoglycans isolated from 1-ml blood samples were assayed with the following techniques: carbazole, electrophoretic and enzymatic. Separation and assay of particular GAG were made with bidirectional electrophoresis. Isomers of the remaining chondroitin sulphates were assayed enzymatically. Electrophoretograms of GAG in blood serum of healthy women have shown two fractions: low sulphate chondroitin sulphate and chondroitin-4-sulphate. The same fractions of GAG were found in blood serum of the female patients with hyperthyroidism. Mean concentration of GAG in the blood serum of hyperthyroid patients increased by 51%: low sulphate chondroitin sulphate and chondroitin-4-sulphate concentrations increased by 22% and 190% respectively. Chondroitin sulphates in the blood serum of both groups were degraded to unsaturated disaccharides not containing sulphur and unsaturated 4-sulphate disaccharides. Concentrations of unsaturated 4-sulphate and unsaturated sulphur-free disaccharides increased by 71% and 17% in hyperthyroidism. Observed changes in the blood serum GAG concentrations reflect changes in the connective tissue metabolism in hyperthyroidism.

Urinary glycosaminoglycans in patients with hypothyroidism and in healthy subjects.

Although the changes in urinary glycosaminoglycans have been investigated in several endocrinopathies, no information was hitherto available on the content and composition of urinary glycosaminoglycans in hypothyroidism. Urinary glycosaminoglycans were therefore investigated in patients with hypothyroidism and in healthy subjects. The total daily excretion of urinary glycosaminoglycans was found to be significantly increased (by 41%) in hypothyroidism. Two electrophoretic bands were always detected in both examined groups: a major band of chondroitin sulphate and a minor band of heparan sulphate. Heparan sulphate and chondroitin sulphate levels were respectively 114% and 42% higher in patients with hypothyroidism than in controls. The respective increases in chondroitin-4-sulphate and chondroitin-6-sulphate were 31% and 41%. The relative quantities of chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate and non-sulphated chondroitin sulphate were unchanged in the two examined groups. The changes observed in the levels of the excreted glycosaminoglycans may reflect the altered metabolism of connective tissue in hypothyroidism.

Biosynthesis of heparin. Purification of a 110-kDa mouse mastocytoma protein required for both glucosaminyl N-deacetylation and N-sulfation.

The polymer modification process in the biosynthesis of heparin/heparan sulfate is initiated by N-deacetylation, followed by N-sulfation, of N-acetylglucosamine units. Chromatography of a detergent extract from mouse mastocytoma on wheat germ agglutinin-Sepharose yielded a protein fraction, eluted with 0.3 M N-acetylglucosamine, that expressed N-deacetylase activity, but only after recombination with proteins that did not bind to the lectin column. In subsequent purification of the active lectin-bound component, all assays were performed following addition of the unbound protein fraction. After two additional chromatography steps, on blue Sepharose and 3',5'-ADP-agarose, the lectin-binding N-deacetylase component had been purified about 4300-fold with an 11% yield and showed essentially a single band, corresponding to an apparent molecular weight of approximately 110,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the purified 110-kDa protein showed that it contained, in addition to the N-deacetylase, N-sulfotransferase activity; however, the expression of N-sulfotransferase activity was independent of additional proteins. Backtracking the N-sulfotransferase through the purification scheme previously applied to the N-deacetylase showed the two enzyme activities to the N-deacetylase showed the two enzyme activities to be cofractionated in each separation step. It is proposed that the expression of glucosaminyl N-deacetylase activity depends on the concerted action of (at least) two protein components, one of which also possesses glucosaminyl N-sulfotransferase activity.

Biosynthesis of heparin. Different molecular forms of O-sulfotransferases.

O-Sulfotransferases involved in heparin biosynthesis were purified > or = 10,000-fold from detergent extracts of mouse mastocytoma tissue by sequential chromatographies on DEAE-Sephacel, heparin-agarose, blue Sepharose, and 3',5'-ADP-Sepharose. The resultant preparation catalyzed the transfer of 35S from 3'-phosphoadenosyl-5'-phospho-[35S]sulfate into N,O-desulfated, re-N-sulfated heparin. Anion-exchange high performance liquid chromatography of disaccharides obtained by deaminative cleavage of the 35S-labeled polysaccharide product revealed O-35S-sulfation at C-2 of L-iduronic acid and at C-6 of D-glucosamine units. SDS-polyacrylamide gel electrophoresis of semipurified enzyme followed by extraction of gel segments and renaturation of proteins consistently showed two distinct fractions of O-sulfotransferase activity, corresponding to proteins of approximately 20 and approximately 60 kDa. The approximately 60-kDa enzyme(s) catalyzed both the 2-O- and 6-O-sulfotransferase reactions, whereas the approximately 20-kDa fraction promoted iduronosyl 2-O-sulfation only. These results are discussed in relation to previous findings, indicating that some of the enzymes involved in heparin biosynthesis catalyze more than one reaction.

Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli.

We report a rapid, large-scale process for the purification of a recombinant Fab fragment specific for the tobacco mosaic virus coat protein (Fab57P). The fragment is expressed periplasmically in Escherichia coli. The expression level was optimized in 0.3-L fermentors. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degrees C), (2) combined use of two beta-lactam antibiotics (carbenicillin and ampicillin), (3) IPTG concentration 0.1 mM, (4) regulated pH 7.2, (5) 17-h induction time, and (6) conditions that reduce mechanical stress. Optimized large-scale fermentations were done in 15- and 300-L capacity fermentors. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the crude E. coli homogenate was directly applied, without centrifugation, to an SP Sepharose Big Beads column. The recombinant Fab fragment was eluted as a single peak in a sodium chloride gradient. The fragment was further purified by affinity adsorption to a column packed with Epoxy-activated Sepharose 6B to which the antigen peptide NH(2)-CGS YNR GSF SQS SGLV-CONH(2) had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%.