Structure of the catalytic domain of human protein kinase C beta II complexed with a bisindolylmaleimide inhibitor.

The conventional protein kinase C isoform, PKCII, is a signaling kinase activated during the hyperglycemic state and has been associated with the development of microvascular abnormalities associated with diabetes. PKCII, therefore, has been identified as a therapeutic target where inhibitors of its kinase activity are being pursued for treatment of microvascular-related diabetic complications. In this report, we describe the crystal structure of the catalytic domain of PKCbetaII complexed with an inhibitor at 2.

The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme.

NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions.

Structure-function analysis of the mammalian DNA polymerase beta active site: role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer.

The crystal structure of the catalytic domain of rat DNA polymerase beta revealed that Asp256 is located in proximity to the previously identified active site residues Asp190 and Asp192. We have prepared and kinetically characterized the nucleotidyl transfer activity of wild type and several mutant forms of human and rat pol beta. Herein we report steady-state kinetic determinations of KmdTTP, Km(dT)16, and kcat for mutants in residue Asp256 and two neighboring residues, Arg254 and Arg258, all centrally located on strand beta 7 in the pol beta structure.

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain.

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently in Escherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved in de novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed in E.

Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase.

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer.

Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase.

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity.

Synthesis and secretion of serum gelsolin by smooth muscle tissue.

Gelsolin is one of many actin binding proteins which regulate the structure of intracellular microfilaments. A secretory form of gelsolin, a protein also known as "actin depolymerizing factor" or "brevin," is present in animal sera. In the present studies, we: demonstrate that a 90-kDa secretory protein produced by chicken gizzard smooth muscle is serum gelsolin; show that chicken serum gelsolin, as compared with its mammalian counterparts, lacks 26 amino acid residues at its NH2-terminal end; show that gizzard smooth muscle devotes on the order of 100 times more of its total protein synthetic effort (about 1% of the total) to the production of serum gelsolin than does liver, a previously speculated major source of this protein; and give evidence that rat tissues which are rich in smooth muscle cells (blood vessels, uterine muscle) also produce serum gelsolin.

Cycloheptatrienylidene analog of 11-cis retinal. Formation of pigment in photoreceptor membranes.

In biochemical and electrophysiologic studies employing the bullfrog (Rana catesbeiana) and the rat, the authors examined the interaction of opsin and an 11-cis-locked analog of retinal. In previously bleached preparations of bullfrog receptor outer segments (ROS) and isolated retinas, incubation with the aldehyde form (I) of the analog leads to the appearance of a pigment that is degraded slowly by hydroxylamine but is relatively resistant to photolysis. In the ROS preparation, the analog pigment (lambda max of difference spectrum congruent to 497 nm) also forms on incubation with NADP+ and the alcohol form (II) of the analog.

Utilization of retinoids in the bullfrog retina.

The capacity to generate 11-cis retinal from retinoids arising naturally in the eye was examined in the retina of the bullfrog, Rana catesbeiana. Retinoids, co-suspended with phosphatidylcholine, were applied topically to the photoreceptor surface of the isolated retina after substantial bleaching of the native visual pigment. The increase in photoreceptor sensitivity associated with the formation of rhodopsin, used as an assay for the appearance of 11-cis retinal in the receptors, was analyzed by extracellular measurement of the photoreceptor potential; in separate experiments using the isolated retina or receptor outer segment preparations, the formation of rhodopsin was measured spectrophotometrically.