Electrophoretic investigations of the acid conformational change of alpha-lactalbumin.

In order to clarify how the electrophoretic behavior reflects the conformational transition of globular proteins, moving boundary electrophoresis was applied to analysis of the acid conformational change of alpha-lactalbumin. The appearance of only a single electrophoretic boundary in the transition region of the protein suggests a very rapid transition with a half-time estimated to be smaller than 7 min on the basis of the theory of isomerizing systems in electrophoresis. The transition is clearly reflected in the dependence of the mobility on the protein net charge, which shows a sigmoidal curve closely similar to that obtained by a Linderstr?m-Lang pH-tritration plot for the carboxyl groups of alpha-lactalbumin. It was also concluded from the transition curves that the acidfication does not result in complete unfolding, but that a compact structure is maintained in the acidic region with an apparently expanded form as compared to the native state of the protein. All results obtained by electrophoresis were also supported by the results of pH-jump studies, analytical gel chromatography, and CD measurements.     

Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin

To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.     

In Vitro Proliferative Response and Polyclonal Antibody Production in Spleen Cells of Immunologically Defective CBA/N and C3H/HeJ Mice by Water-Soluble Adjuvant (Bu-WSA) Extracted from Bacterionema matruchotii

Mitogenicity and the polyclonal plaque forming cell(PFC)-inducing property of a water soluble-adjuvant extracted from Bacterionema matruchotii by butanol (Bu-WSA) were examined in vitro in the spleen cells of hybrid (CBA/N female × BALB/c male) F1 mice and C3H strain of mice. The hybrid F1 male cells which expressed a CBA/N-defect were unable to respond to Bu-WSA, when assessed by the incorporation of [³H] thymidine into the cells and the generation of anti-trinitrophenyl (TNP)-PFC or autoantibody PFC defined by the anti-bromel-ain-treated mouse erythrocyte PFC assay. However, hybrid F1 female cells with normal traits responded to Bu-WSA. Cultured spleen cells of bacterial lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mice responded to Bu-WSA as in the case of cells of LPS-responsive C3H/He mice, and the [³H]thymidine-uptakes and the numbers of PFC in these culture cells increased. Re-extraction of Bu-WSA by phenol did not affect its activities, while the activity of butanol-extracted LPS on C3H/HeJ cells decreased after re-extraction by the same procedure with phenol.     

Testing of SHIRPA, a mouse phenotypic assessment protocol, on Dmd mdx and Dmd mdx3cv dystrophin-deficient mice

The SHIRPA protocol was proposed as a rapid, comprehensive screening method for qualitatively abnormal phenotypes in the mouse (Rogers et al., Mamm Genome 8, 711, 1997). This screening technique is currently being used to identify mutants induced by N-ethylnitrosourea (ENU) mutagenesis (Brown and Nolan, Hum Mol Genet 7, 1627, 1998). SHIRPA can be used to identify mutants with neuromuscular abnormalities, but the sensitivity of the protocol is unknown. We tested two dystrophin-deficient mutants Dmd ^mdx and Dmd ^mdx3cv , both of which are indistinguishable from wild-type by a simple visual assessment, at different ages, using the primary screen of the SHIRPA protocol. The most dramatic observation was that both Dmd ^mdx and Dmd ^mdx3cv mice showed extreme fatigue after testing, while mice from the same C57BL strains appeared unaffected. Each strain of dystrophin-deficient mice showed a different profile in locomotor activity and deficiencies in the wire maneuver, righting reflex, and negative geotaxis tests. Furthermore, the wire maneuver test indicated an earlier onset of muscular impairment in Dmd ^mdx than Dmd ^mdx3cv mice. These data suggest that the SHIRPA primary screen is effective not only in identifying subtle neuromuscular mutants, but also in distinguishing qualitative differences between mutants with neuromuscular abnormalities. Received: 5 August 1999 / Accepted: 14 April 2000     

Polymer concentration dependence of the helix to random coil transition of a charged polypeptide in aqueous salt solution.

The helix to coil transition of poly(L-glutamic acid) was investigated in 0.05 and 0.005 M aqueous potassium chloride solutions by use of potentiometric titration and circular dichroism measurement. Polymer concentration dependence of the transition was observed in the range from 0.006 to 0.04 monomol/e in 0.005 M KG1 solution. The polymer concentration dependence can be interpreted by current theories of the transition of charged polypeptides and of titration curves of linear weak polyelectrolytes taking the effect of polymer concentration into consideration.     

Isolation of protoplasts from somatic embryos of carrot

Protoplasts were isolated enzymatically from synchronously induced globular somatic embryos from a carrot suspension culture. Among the macerating enzymes tested, Driselase was the most effective for release of protoplasts from embryos. A higher medium osmolarity was required for the isolation of protoplasts from embryos than from undifferentiated cells. Protoplasts from embryos were smaller than protoplasts from undifferentiated cells. On step gradients of Ficoll, protoplasts from embryos gave one major band. Protoplasts from undifferentiated cells gave two major bands, one lighter and the other heavier than the protoplasts from embryos.