Pin1 is a prolyl isomerase that recognizes phosphorylated Ser/Thr-Pro sites and phosphatase inhibitor-2 (I-2) is phosphorylated during mitosis at a PSpTP site that is expected to be a Pin1 substrate. However, we previously discovered I-2, but not phospho-I-2, bound to Pin1 as an allosteric modifier of Pin1 substrate specificity (Li et al., Biochemistry 47: 292, 2008). Here, we used binding assays and NMR spectroscopy to map the interactions on Pin1 and I-2 to elucidate the organization of this complex. Despite only ~ 50% sequence identity, human, Xenopus and Drosophila I-2 proteins all exhibited identical, saturable binding to GST-Pin1 with K0.5 of 0.3 μM. The [1H-15N] Heteronuclear Single Quantum Coherence spectra for both the WW domain and isomerase domain of Pin1 showed distinctive shifts upon addition of I-2. Conversely, in NMR spectroscopy specific regions of I-2 were affected by addition of Pin1. A single residue substitution I68A in I-2 reduced binding to Pin1 by half and essentially eliminated binding to the isolated WW domain. On the other hand truncation of I-2 to residue 152 had minimal effect on binding to WW domain but eliminated binding to the isomerase domain. Size exclusion chromatography revealed that wild type I-2 plus Pin1 formed a large (> 300 kDa) complex, whereas I-2(I68A) formed a complex of half the size that we propose are a heterotetramer and a heterodimer, respectively. Pin1 and I-2 are conserved among eukaryotes from yeast to human, and we propose they are an ancient partnership that provides a means to regulate Pin1 specificity and function.

The alpha-galactosidases in normal man-Chinese hamster somatic cell hybrids were investigation with antibodies specific for human alpha-galactosidase A and antibodies specific for Chinese hamster alpha-galactosidase. It was found that an isoenzyme in hybrid cells, which has an electrophoretic mobility between that of human alpha-galactosidase A and Chinese hamster alpha-galactosidase, contains immunologic determinants of both human and Chinese hamster origin, suggesting that it is a heteropolymeric molecule. Moreover, the locus for human alpha-galactosidase, which was found to be X-linked, is the locus coding for alpha-galactosidase A. Hybrids isolated after fusion of Chinese hamster cells with cells of a patient with Fabry's disease did not express human alpha-galactosidase A or the heteropolymeric molecule even in the presence of the active human X chromosome, indicating that the deficiency of alpha-galactosidase A in Fabry's disease is probably due to a mutation in a structural gene resulting in the inability to form immunologically detectable and functionally active molecules of alpha-galactosidase A.

Quantitative estimations of glutathione S-transferase activities with 1-chloro-2,4-dinitrobenzene as the electrophilic second substrate, in 142 postmortem human tissue specimens derived from 34 different organs of one or more of 13 individuals belonging to various age groups, are presented. Collectively the data indicate: (1) all tissues examined have appreciable levels of enzyme activity; (2) liver, kidney, lung, muscle, heart, adrenal glands, pancreas, and stomach of fetal origin possess higher enzyme activities than those of the adults, and (3) there are wide interindividual variations in the tissue enzyme activities.

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