The LIM motif is a cysteine- and histidine-rich sequence that was first identified in proteins involved in control of gene expression and cell differentiation. In order to characterize structural features of the LIM domain, we have carried out biophysical studies on two polypeptides that display LIM domains: the cysteine-rich intestinal protein (CRIP) and a fragment of the cysteine-rich protein (CRP). Bacterial expression vectors were constructed for the intact CRIP molecule and the C-terminal half of CRP, designated LIM2, such that each expressed protein contained a single LIM domain. Both proteins were recovered as soluble, Zn(II)-containing proteins. The metal coordination properties of these two distinct LIM domain proteins were highly similar, suggesting that a common structural architecture may exist in LIM domain proteins. Both proteins exhibit a maximum of two tetrahedrally bound Zn(II) ions per molecule. Electronic spectroscopy of Co(II) complexes and 113Cd NMR of Cd(II) complexes of CRIP and LIM2 revealed a similar ligand field pattern with one tetrathiolate (S4) site and one S3N1 site for divalent metal ions. The nitrogen ligand was shown to arise from a histidyl imidazole by heteronuclear multiple quantum coherence NMR. The eight conserved residues within the LIM domains of CRIP and LIM2 include seven cysteines and one histidine. It is likely that these conserved residues generate the S4 and S3N1 Zn(II)-binding sites. Metal binding to the two sites within a single LIM domain is sequential, with preferential occupancy of the S4 site. Slow metal ion exchange occurs between sites within an LIM domain, and metal exchange with exogenous metal ions is observed, with exchange at the S3N1 site being kinetically more facile. In the absence of metal binding both proteins appear to be substantially unfolded. Metal binding stabilizes a tertiary fold containing appreciable secondary structural elements. The common metal ion coordination in CRIP and LIM2 suggests that the LIM motif may constitute a structural module with conserved features.

No abstract

No abstract

In vivo 19F NMR at 4.7 T has shown that the biphasic elimination of the vapor anesthetic isoflurane from rat brain is ca 15% slower in old (23-24 months) animals compared with young (5-6 months) animals. The fast kinetic component has a t1/2 of ca 7-9 min and the slow event, 100-115 min. Gas chromatographic measurement of arterial blood elimination displays age attenuation to the same extent, although a monophasic kinetic process (6-7 min). The slow wash-out from brain is thought to involve elimination from intracranial fatty tissue as postulated by others in rabbit brain. Longitudinal relaxation time measurements show monoexponential recovery and essentially identical values for young (1.09 + 0.11 s) and old (1.04 +/- 0.09 s) animals. For dipalmitoylphosphatidylcholine vesicles the monoexponential recovery also suggests rapidly exchanging averaged homogeneous lipid environments for the anesthetic, but the longer T1s (2.75 +/- 0.25 s) imply less restricted mobility compared with brain. Single T2 values were obtained in vivo, indicating either a single compartment or rapid exchange between multiple environments. These measurements were inconsistent, undoubtedly as a result of B1 inhomogeneity. The age-attenuated elimination kinetics for isoflurane are consistent with poorer cardiopulmonary function, whereas the T1 data suggest similar environments for the anesthetic in young and old brain tissue.

The whole blood pharmacokinetics of intravenously administered 99mTc-disofenin (DISIDA) have been studied in dogs. Serial blood sampling permitted calculation of whole blood disposition rates, which principally represent liver clearance. There were striking differences in these rates between 6 normals and 7 animals in whom liver damage was induced by chronic bile duct ligation (256 vs 58 ml/min, P less than 0.001). Blood levels of radioactivity fell in a biexponential fashion characterized by rapid and slow disposition phases, whose half times were 2.4 and 58 min in normal animals. On 3 occasions, plasma was obtained from 1 animal by exsanguination 35 min after the administration of DISIDA and rapidly transfused into a 2nd animal. The whole blood pharmacokinetics of the second (recipient) animal showed a predominance of the slow disposition phase and a small rapid phase. The hepatic extraction ratio of blood radioactivity was measured in 3 dogs and was high (75%-90%) early after injection of DISIDA, but fell rapidly to remain around 10%. These experiments suggest the presence of two different species in the radiopharmaceutical studied, each being removed from the blood stream by the liver, but at different rates. The contribution of renal clearance to overall whole blood pharmacokinetics was negligible, since three nephrectomized dogs displayed similar pharmacokinetics to normals. Whole blood DISIDA pharmacokinetics are more complex than previously thought but appear to be capable of providing an accurate measure of liver function.