Membrane ultrafiltration: concentration of interleukin-3.

A pilot-plant-scale operation was used for studying membrane ultrafiltration and concentration of kiloliter quantities of the lymphokine interleukin-3 with a single set of membranes. Initial use of ammonium sulfate precipitation of interleukin-3 proved erratic in the recovery of biological activity and resulted in corrosion of the processing equipment. Membrane ultrafiltration proved to be effective in enabling control of the degree of concentration and predicting recovery of the biologically active protein.     

H3-rules: identification of CDR-H3 structures in antibodies.

For the third complementarity determining region of the antibody heavy chain (CDR-H3), we propose the 'H3-rules', which should identify the tertiary structure from the amino acid sequence of the CDR-H3 segment. A total of 100 CDR-H3 segments from well-determined crystal structures were analyzed. Distinctive relationships between the structures and the sequences were revealed from 55 segments, and the rules were examined for the other 45 segments and were verified. In some antibodies, basic residues at specific positions were revealed to be notable signals, with their ability to form salt bridges and to assume conformations inconsistent with the rules.     

Haemonchus contortus: enzymes. 3. Glutamate dehydrogenase

Adult male and female Haemonchus contortus were homogenized and subjected to differential centrifugation. The crude, high-speed, supernatant fraction contained more than 95% of the glutamate dehydrogenase activity. The enzyme was purified through use of DEAE-cellulose columns and sucrose density gradient centrifugation. The enzyme from both crude and purified preparations was detected as a single band of activity following starch or polyacrylamide-gel electrophoresis. The Haemonchus enzyme was compared with ovine and bovine liver glutamate dehydrogenases. The three enzymes were similar in molecular size, Michaelis constants, and pH optimums but differed in electrophoretic mobility in polyacrylamide-gels, activity with NADP as coenzyme, and effect of AMP and ADP on activity. Sheep anti-Haemonchus glutamate dehydrogenase serum inhibited Haemonchus glutamate dehydrogenase, but did not inhibit the ovine or bovine enzymes.     

3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. An optimized assay in homogenates of developing brain.

An optimized in vitro assay of 3'-phosphoadenylysulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11, galactosylceramide sulfotransferase, formerly known as galactocerebroside sulfotransferase) activity is presented, that can be used in crude homogenate of brain tissue of various developmental stages. The enzyme activity is determined by measuring the [35S]sulfatides formed by the enzymic transfer of [35S]sulfate from 3'-phosphoadenoside 5'-phospho [35S]sulfate to galactosylceramides. The sulfatide formation at 30 degrees C is linear up to 30 min and up to a protein concentration of 1 mg per 0.5 ml assay volume. The presence of 0.4% Triton X-100 and 50 micrometer exogenous bovine cerebrosides are optimal for enzyme activity. The pH optimum of the reaction is at pH 6.5 using 0.1 M imidazole buffer. The enzyme reaction is stimulated by NaCl, KCl, MgCl2, CaCl2, MnCl2, ATP and inhibited by ADP. The developmental enzyme activity pattern of mouse brain is the same, if derived from homogenates and microsomes, respectively, under our assay conditions.