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The hydrolysis of isolated β-lactoglobulin (9 and 70−200 mg/mL) by a Bacillus licheniformis protease was followed to assess whether aggregates and gels, respectively, were formed during hydrolysis. Changes during hydrolysis were monitored by electrophoresis, dynamic light scattering, and fluorescence and circular dichroism spectroscopy. Gelation was monitored by dynamic oscillation rheology. Upon hydrolysis of a β-lactoglobulin preparation with the B. licheniformis protease aggregates were formed and a soft gel resulted from only 70 mg/mL of β-lactoglobulin. The aggregates consisted of a number of peptides with molecular weight ranging from 2000 to 6000 and pI from 5 to 8. As the aggregates were solubilized in either SDS or urea or at extreme pH values, it is proposed that noncovalent interactions, mainly electrostatic and hydrophobic, are major interacting forces. These kinds of aggregates are thought to be important in protease-induced gelation of whey protein isolate solutions. Keywords: β-Lactoglobulin; proteolysis; aggregation; fluorescence; circular dichroism

ATP7A encodes a copper-translocating ATPase that belongs to the large family of P-type ATPases. Eight conserved regions define the core of the P-type ATPase superfamily. We report here the identification of 21 novel missense mutations in the conserved part of ATP7A that encodes the residues p.V842-p.S1404. Using the coordinates of X-ray crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase, as determined in the presence and absence of Ca(2+), we created structural homology models of ATP7A. By mapping the substituted residues onto the models, we found that these residues are more clustered three-dimensionally than expected from the primary sequence. The location of the substituted residues in conserved regions supports the functional similarities between the two types of P-type ATPases. An immunofluorescence analysis of Menkes fibroblasts suggested that the localization of a large number of the mutated ATP7A protein variants was correct. In the absence of copper, they were located in perinuclear regions of the cells, just like the wild type. However, two of the mutated ATP7A variants showed only partly correct localization, and in five cultures no ATP7A protein could be detected. These findings suggest that although a disease-causing mutation may indicate a functional significance of the affected residue, this is not always the case.

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Native carboxypeptidase A has been crystallized in a new crystal form, and the structure has been refined with X-ray data to 2.0 A resolution. In contrast to the previously published structure [Rees, D. C., Lewis, M., and Lipscomb, W. N. (1983) J. Mol. Biol. 168, 367-387], no active-site amino acids are involved in the crystal packing. The important Tyr248 is stabilized inside the active site by a hydrogen bond and by interactions with Ile247. The proposed role of Tyr248 in the induced fit mechanism is therefore not supported by the findings in this structure of native carboxypeptidase A. The structure has a partly populated inhibitory Zn2+ site in close proximity to the catalytic Zn2+ as evident from X-ray anomalous dispersion data. A hydroxo bridge is found between the catalytic Zn2+ and the inhibitory Zn2+ with a Zn2+-Zn2+ distance of 3.48 A. In addition, the inhibitory Zn2+ has Glu270 as a monodentate ligand. No other protein ligands to the inhibitory Zn2+ are seen. The crystals were grown at 0.3 M LiCl and weak evidence for a binding site for partly competitive inhibitory anions is observed.

We have used the 2.6 A î structure of the rabbit sarcoplasmic reticulum Ca 2+ -ATPase isoform 1a, SERCA1a [Toyoshima, C., Nakasako, M., Nomura, H. + , suggesting a previously unknown mechanism for transport of protons. Comparison with the structure of the SERCA1a made it feasible to suggest a possible receptor region for the C-terminal auto-inhibitory domain extending from the phosphorylation and anchor domains into the transmembrane region. ß

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