Glucose is the main physiological stimulus for insulin biosynthesis and secretion by pancreatic ␤-cells. Glucose-6-phosphatase (G-6-Pase) catalyzes the dephosphorylation of glucose-6-phosphate to glucose, an opposite process to glucose utilization. G-6-Pase activity in pancreatic islets could therefore be an important factor in the control of glucose metabolism and, consequently, of glucose-dependent insulin secretion. While G-6-Pase activity has been shown to be present in pancreatic islets, the gene responsible for this activity has not been conclusively identified. A homolog of liver glucose-6-phosphatase (LG-6-Pase) specifically expressed in islets was described earlier; however, the authors could not demonstrate enzymatic activity for this protein. Here we present evidence that the previously identified islet-specific glucose-6-phosphatase-related protein (IGRP) is indeed the major islet glucose-6-phosphatase. IGRP overexpressed in insect cells possesses enzymatic activity comparable to the previously described G-6-Pase activity in islets. The K m and V max values determined using glucose-6-phosphate as the substrate were 0.45 mM and 32 nmol/mg/min by malachite green assay, and 0.29 mM and 77 nmol/mg/min by glucose oxidase/peroxidase coupling assay, respectively. High-throughput screening of a small molecule library led to the identification of an active compound that specifically inhibits IGRP enzymatic activity. Interestingly, this inhibitor did not affect LG-6-Pase activity, while conversely LG-6-Pase inhibitors did not affect IGRP activity. These data demonstrate that IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors to this molecule can be obtained. IGRP inhibitors may be an attractive new approach for the treatment of insulin secretion defects in type 2 diabetes.

Using subfragments of the simian virus 40 (SV40) core origin, we demonstrate that two alternative modules exist for the assembly of T-antigen (T-ag) double hexamers. Pentanucleotides 1 and 3 and the early palindrome (EP) constitute one assembly unit, while pentanucleotides 2 and 4 and the AT-rich region constitute a second, relatively weak, assembly unit. Related studies indicate that on the unit made up of pentanucleotide 1 and 3 and the EP assembly unit, the first hexamer forms on pentanucleotide 1 and that owing to additional protein-DNA and protein-protein interactions, the second hexamer is able to form on pentanucleotide 3. Oligomerization on the unit made up of pentanucleotide 2 and 4 and the AT-rich region is initiated by assembly of a hexamer on pentanucleotide 4; subsequent formation of the second hexamer takes place on pentanucleotide 2. Given that oligomerization on the SV40 origin is limited to double-hexamer formation, it is likely that only a single module is used for the initial assembly of T-ag double hexamers. Finally, we discuss the evidence that nucleotide hydrolysis is required for the remodeling events that result in the utilization of the second assembly unit.A thorough understanding of the initiation of DNA replication and its regulation, will require a detailed description of the protein-DNA and protein-protein interactions that take place at origins of replication. Since origins of replication in higher eukaryotic organisms are poorly characterized (8, 21), little is known about the molecular interactions that take place at these sites. Therefore, well-defined viral model systems are being used in an effort to establish the molecular interactions required to initiate DNA replication. One of the best-characterized viral model systems is that based on simian virus 40 (SV40). This virus encodes a single protein, termed T antigen (T-ag) (68), that binds in a site-specific manner to the viral origin of replication, a necessary step for the initiation of DNA replication (72). Several reviews have been published that cover the SV40 origin of replication, T-ag, and the interactions that take place between these molecules (4,7,26). Therefore, only a brief introduction is provided that stresses recent observations in this field.A 64-bp region of the SV40 genome, termed the core origin, is necessary and sufficient for viral replication (19,22,39,52,66). The core origin consists of a central region, termed site II, that is flanked by an AT-rich domain (AT) and a second region, termed the early palindrome (EP) (17). Site II contains four GAGGC pentanucleotides, arranged as inverted pairs, that serve as binding sites for T-ag (20,43,69,71). All three regions of the core origin are required for DNA unwinding and initiation of DNA replication (13,17,32).T-ag is a 708-amino-acid phosphoprotein that contains several structural and functional domains (for reviews, see references 7 and 26). One domain of T-ag, the T-ag origin binding domain (T-ag-obd 131-260 ) has been extensively studied. The purified T-a...

Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr3Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed.Initiation of DNA replication is a complicated and highly regulated process that takes place during the S phase of the cell cycle. Progress in understanding initiation events in eukaryotes includes the identification of many of the factors that catalyze nascent DNA synthesis (reviewed in references 6, 7, 19, 32 and 90). Moreover, the isolation of the origin replication complex (ORC) (2) and related factors (reviewed in references 22 and 90) has provided considerable insight into initiation of eukaryotic DNA replication. However, since origins of replication from higher eukaryotes have not been characterized (8,19), much remains to be learned about the protein-DNA interactions that are responsible for the initiation of DNA replication in higher organisms.Experiments conducted with viral model systems have overcome certain of these limitations and aided in efforts to understand the molecular interactions that are necessary to initiate DNA replication in eukaryotes (19). In several instances, the sequences that define viral origins of replication have been established and the protein-DNA interactions that take place at these sequences have been extensively characterized (19). One particularly useful viral model system is based on simian virus 40 (SV40) DNA replication in vitro (43,83,95). SV40 encodes an 82-kDa protein, termed T antigen (T-ag) (84), that plays a number of critical roles during initiation of DNA replication. The functions of T-ag during the initiation of viral DNA replication have been the topic of several reviews (4, 7, 26). Briefly, T-ag site specifically binds to the SV40 origin of replication as a monomer and, as a result of a series of additional protein-protein and protein-DNA interactions (reviewed in references 4 and 7), oligomerizes into a double hexamer (13,15,51,71). The double hex...

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd131–260) have been determined. Binding of the purified T-ag-obd131–260 is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.

Inositol-specific PLCs comprise a family of enzymes that utilize phosphoinositide substrates, e.g., PIP(2), to generate intracellular second messengers for the regulation of cellular responses. In the past, monitoring this reaction has been difficult due to the need for radiolabeled substrates, separation of the reaction products by organic-phase extraction, and finally radiometric measurements of the segregated products. In this report, we have studied the enzymatic characteristics of two novel PLCs that were derived from functional genomic analyses using a phospholipid-modified solid scintillating support. This method allows for the hydrophobic capture of the [(3)H]phosphoinositide substrate on a well defined scintillation surface and the homogenous measurement of the enzymatic hydrolysis of the substrate by proximity effects. Our results show that the assay format is robust and well suited for this class of lipid-metabolizing enzymes.