Biomedical subjects

J D Hurt

Publications and source records attributed to J D Hurt.

4 recordsLinked to original sources

PubMed1998

Structures of murine carbonic anhydrase IV and human carbonic anhydrase II complexed with brinzolamide: molecular basis of isozyme-drug discrimination.

Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.

Amino Acid Sequence↗

PubMed1998

Isolation and expression of murine carbonic anhydrase IV.

A 1193-bp cDNA containing the complete murine carbonic anhydrase IV coding sequence was isolated from a Balb/c kidney cDNA library. The entire coding sequence plus shorter segments was used in an Escherichia coli T7 expression vector system to produce four forms of murine CA IV, including (1) a protein representing the full-length coding sequence, (2) an amino-truncated protein lacking the 18 N-terminal amino acid plasma membrane targeting sequence, (3) a protein which lacked the plasma membrane targeting sequence and 26 C-terminal amino acids, and (4) a protein which lacked both 36 N-terminal residues (the plasma membrane targeting sequence plus 18 additional amino acids which included the first two cysteines) and 26 C-terminal residues. All four proteins were expressed as catalytically inactive inclusion bodies. After rapid dilution of washed, guanidine hydrochloride-denatured inclusion bodies into a glutathione-, l-arginine-containing renaturation buffer, an active carbonic anhydrase IV at yields of 3-4 mg/liter was easily purified from cultures expressing the form lacking the N-terminal targeting sequence and 26 C-terminal residues. The longest and shortest forms of carbonic anhydrase IV failed to refold into active enzyme under these conditions. The activity of purified recombinant carbonic anhydrase IV was highly resistant to sodium dodecyl sulfate, as is the native enzyme. This resistance presumably results from intramolecular disulfide bonds maintaining a functional active site configuration even in the presence of denaturing agents.

Amino Acid Sequence↗

PubMed1997

Catalytic properties of murine carbonic anhydrase IV.

A cDNA encoding the murine carbonic anhydrase IV (mCA IV) gene, modified to resemble a form of mature human carbonic anhydrase IV (Okuyama, T., Waheed, A., Kusumoto, W., Zhu, X. L., and Sly, W. S. (1995) Arch. Biochem. Biophys. 320, 315-322), was expressed in Escherichia coli. Inactive inclusion bodies were collected and refolded, and active enzyme was purified; the resulting mCA IV was used to characterize the catalysis of CO2 hydration using stopped flow spectrophotometry and 18O exchange between CO2 and water. Unlike previously studied isozymes in this class of carbonic anhydrase, the pH profile for kcat for hydration of CO2 catalyzed by mCA IV could not be described by a single ionization, suggesting multiple proton transfer pathways between the zinc-bound water molecule and solution. A role for His64 in transferring protons between the zinc-bound water and solution was confirmed by the 100-fold lower activity of the mutant of mCA IV containing the replacement His64 --> Ala. The remaining activity in this mutant at pH levels near 9 suggested a second proton shuttle mechanism. The maximal turnover number kcat for hydration of CO2 catalyzed by mCA IV was 1.1 x 10(6) s-1 at pH > 9. A pKa of 6.6 was estimated for the zinc-bound water molecule in mCA IV.

Amino Acid Sequence↗

PubMed1988

CPF-550 vs. C-Lite: a comparison study.

We conducted a two-part study to evaluate patient satisfaction with the Corning CPF-550 lenses and to determine whether or not the success of these lenses can be attributed to their selective attenuation of the short wave-lengths of light. In the first part of our investigation we asked nine low vision patients who were currently wearing CPF-550 lenses to evaluate the performance of the tint. Their responses were generally very favorable. In the second part of the study, seven of those patients were given identical prescriptions made of C-Lite material with a solid gray tint on the back surface and a photo-gray extra front surface. They were asked to compare the two filters. To our surprise, six of the seven preferred the more neutral density C-Lite lenses over the CPF-550 filter. Although we have been encouraged by the results of the first part of the study to continue prescribing the CPF-550 lenses, the second part of the study has caused us to question the theory behind the success of these lenses and has alerted us to the fact that some individuals may be even more satisfied with a neutral gray photochromic tint.

Adult↗