Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis.

Clinical Feasibility of Using Single-isocentre Non-coplanar Volumetric Modulated Arc Therapy Combined with Non-coplanar Cone Beam Computed Tomography in Hypofractionated Stereotactic Radiotherapy for Five or Fewer Multiple Intracranial Metastases.

AIMS: To evaluate the clinical feasibility of single-isocentre non-coplanar volumetric modulated arc therapy (NC-VMAT) with non-coplanar cone beam computed tomography (NC-CBCT) in hypofractionated stereotactic radiotherapy (HSRT) for five or fewer multiple brain metastases. MATERIALS AND METHODS: Ten patients with multiple brain metastases who underwent single-isocentre NC-VMAT HSRT with limited couch rotations (within ±45°) and NC-CBCT with a limited scanning range (150-200°) were included in the current analysis. Conventional single-isocentre coplanar VMAT (C-VMAT) plans were generated and compared with NC-VMAT plans. The intracranial response and toxicities of single-isocentre NC-VMAT HSRT were also evaluated. RESULTS: Compared with C-VMAT, NC-VMAT generated better target conformity (P < 0.05), a lower gradient index (P < 0.05) and better normal brain tissue sparing, especially for volume ≥12 Gy, with a median reduction of 12.65 cm3. For 45° couch rotation, NC-CBCT produced sufficient image quality to differentiate bony anatomy, even with a 150° scanning range, which could be successfully used for patient set-up correction. After NC-CBCT, 57.1% of the measured non-coplanar set-up errors exceeded the threshold value. The median gamma passing rate of NC-VMAT was higher than that of C-VMAT plans (P < 0.05). The non-coplanar beam of NC-VMAT with NC-CBCT corrections exhibited superior gamma passing rate to that without NC-CBCT corrections. The intracranial objective response rate and disease control rate for all patients were 80% (8/10) and 100% (10/10), respectively, and the most common toxicities were headache (20%) and dizziness (20%). CONCLUSION: NC-VMAT with limited couch rotation (within ±45°) combined with NC-CBCT with a limited scanning range (150-200°) markedly improves the plan quality and set-up accuracy in single-isocentre multiple-target HSRT.

Can Optical Surface Imaging Replace Non-coplanar Cone-beam Computed Tomography for Non-coplanar Set-up Verification in Single-isocentre Non-coplanar Stereotactic Radiosurgery and Hypofractionated Stereotactic Radiotherapy for Single and Multiple Brain Metastases?

AIMS: To conduct a direct comparison regarding the non-coplanar positioning accuracy between the optical surface imaging system Catalyst HDTM and non-coplanar cone-beam computed tomography (NC-CBCT) in intracranial single-isocentre non-coplanar stereotactic radiosurgery (SRS) and hypofractionated stereotactic radiotherapy (HSRT). MATERIALS AND METHODS: Twenty patients with between one and five brain metastases who underwent single-isocentre non-coplanar volumetric modulated arc therapy (NC-VMAT) SRS or HSRT were enrolled in this study. For each non-zero couch angle, both Catalyst HDTM and NC-CBCT were used for set-up verification prior to beam delivery. The set-up error reported by Catalyst HDTM was compared with the set-up error derived from NC-CBCT, which was defined as the gold standard. Additionally, the dose delivery accuracy of each non-coplanar field after using Catalyst HDTM and NC-CBCT for set-up correction was measured with SRS MapCHECKTM. RESULTS: The median set-up error differences (absolute values) between the two positioning methods were 0.30 mm, 0.40 mm, 0.50 mm, 0.15°, 0.10° and 0.10° in the vertical, longitudinal, lateral, yaw, pitch and roll directions, respectively. The largest absolute set-up error differences regarding translation and rotation were 1.5 mm and 1.1°, which occurred in the longitudinal and yaw directions, respectively. Only 35.71% of the pairs of measurements were within the tolerance of 0.5 mm and 0.5° simultaneously. In addition, the non-coplanar field with NC-CBCT correction yielded a higher gamma passing rate than that with Catalyst HDTM correction (P < 0.05), especially for evaluation criteria of 1%/1 mm with a median increase of 12.8%. CONCLUSIONS: Catalyst HDTM may not replace NC-CBCT for non-coplanar set-up corrections in single-isocentre NC-VMAT SRS and HSRT for single and multiple brain metastases. The potential role of Catalyst HDTM in intracranial SRS/HSRT needs to be further studied in the future.

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is caused by the deficiency of D-glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Despite both a high incidence and morbidity, the molecular mechanisms underlying this deficiency have eluded characterization. In the present study, the molecular and biochemical characterization of the human G6Pase complementary DNA, its gene, and the expressed protein, which is indistinguishable from human microsomal G6Pase, are reported. Several mutations in the G6Pase gene of affected individuals that completely inactivate the enzyme have been identified. These results establish the molecular basis of this disease and open the way for future gene therapy.

Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is an autosomal recessive inborn error of metabolism caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Southern blot hybridization analysis using a panel of human-hamster hybrids showed that human G6Pase is a single-copy gene located on chromosome 17. To correlate specific defects with clinical manifestations of this disorder, we identified mutations in the G6Pase gene of GSD type 1a patients. In the G6Pase gene of a compound heterozygous patient (LLP), two mutations in exon 2 of one allele and exon 5 of the other allele were identified. The exon 2 mutation converts an arginine at codon 83 to a cysteine (R83C). This mutation, previously identified by us in another GSD type 1a patient, was shown to have no detectable phosphohydrolase activity. The exon 5 mutation in the G6Pase gene of LLP converts a glutamine codon at 347 to a stop (Q347SP). This Q347SP mutation was also detected in all exon 5 subclones (five for each patient) of two homozygous patients, KB and CB, siblings of the same parents. The predicted Q347SP mutant G6Pase is a truncated protein of 346 amino acids, 11 amino acids shorter than the wild type G6Pase of 357 residues. Site-directed mutagenesis and transient expression assays demonstrated that G6Pase-Q347SP was devoid of G6Pase activity. G6Pase is an endoplasmic reticulum (ER) membrane-associated protein containing an ER retention signal, two lysines (KK), located at residues 354 and 355. We showed that the G6Pase-K355SP mutant containing a lysine-355 to stop codon mutation is enzymatically active. Our data demonstrate that the ER protein retention signal in human G6Pase is not essential for activity. However, residues 347-354 may be required for optimal G6Pase catalysis.

Molecular mechanisms of an inborn error of methionine pathway. Methionine adenosyltransferase deficiency.

Methionine adenosyltransferase (MAT) is a key enzyme in transmethylation, transsulfuration, and the biosynthesis of polyamines. Genetic deficiency of alpha/beta-MAT causes isolated persistent hypermethioninemia and, in some cases, unusual breath odor or neural demyelination. However, the molecular mechanism(s) underlying this deficiency has not been clearly defined. In this study, we characterized the human alpha/beta-MAT transcription unit and identified several mutations in the gene of patients with enzymatically confirmed diagnosis of MAT deficiency. Site-directed mutagenesis and transient expression assays demonstrated that these mutations partially inactivate MAT activity. These results establish the molecular basis of this disorder and allow for the development of DNA-based methodologies to investigate and diagnose hypermethioninemic individuals suspected of having abnormalities at this locus.

Mutations in the glucose-6-phosphatase gene are associated with glycogen storage disease types 1a and 1aSP but not 1b and 1c.

Glycogen storage disease (GSD) type 1, which is caused by the deficiency of glucose-6-phosphatase (G6Pase), is an autosomal recessive disease with heterogenous symptoms. Two models of G6Pase catalysis have been proposed to explain the observed heterogeneities. The translocase-catalytic unit model proposes that five GSD type 1 subgroups exist which correspond to defects in the G6Pase catalytic unit (1a), a stabilizing protein (1aSP), the glucose-6-P (1b), phosphate/pyrophosphate (1c), and glucose (1d) translocases. Conversely, the conformation-substrate-transport model suggests that G6Pase is a single multifunctional membrane channel protein possessing both catalytic and substrate (or product) transport activities. We have recently demonstrated that mutations in the G6Pase catalytic unit cause GSD type 1a. To elucidate whether mutations in the G6Pase gene are responsible for other GSD type 1 subgroups, we characterized the G6Pase gene of GSD type 1b, 1c, and 1aSP patients. Our results show that the G6Pase gene of GSD type 1b and 1c patients is normal, consistent with the translocase-catalytic unit model of G6Pase catalysis. However, a mutation in exon 2 that converts an Arg at codon 83 to a Cys (R83C) was identified in both G6Pase alleles of the type 1aSP patient. The R83C mutation was also demonstrated in one homozygous and five heterogenous GSD type 1a patients, indicating that type 1aSP is a misclassification of GSD type 1a. We have also analyzed the G6Pase gene of seven additional type 1a patients and uncovered two new mutations that cause GSD type 1a.

Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14.

We have previously reported on the identification of a cDNA (placenta growth factor, PlGF) coding for a novel angiogenic factor expressed in placental tissue that is similar to vascular permeability factor/vascular endothelial growth factor (VPF/VEGF). Biochemical and functional characterization of PlGF derived from transfected COS-1 cells revealed that it is a glycosylated dimeric secreted protein able to stimulate endothelial cell growth in vitro. Here, we report the isolation and characterization of the PlGF gene located on chromosome 14. At least two different mRNAs are produced from this single-copy gene in different cell lines and tissues. Sequence comparison of the polypeptides encoded by the two different isolated cDNAs indicates that they are identical except for the insertion of a highly basic 21 amino acid stretch at the carboxyl end of the protein. RNA expression analysis of several tissues, tumors and cell lines indicates differential distribution of the two PlGF mRNAs. Finally, preliminary results indicate that the PIGF gene has been conserved in evolution, since the human PlGF cDNA hybridizes to sequences present in the genomic DNA of Drosophila, Xenopus, chicken and mouse.

Immortalization of virus-free human placental cells that express tissue-specific functions.

Human pregnancy-specific glycoproteins (PSGs) are a family of closely related placental proteins that, together with the carcinoembryonic antigen members, comprise a subfamily within the immunoglobulin superfamily. To facilitate study of the control of PSG expression, we immortalized human placental cell lines with adenovirus-origin-minus (ori-)-simian virus-40 (SV40) recombinant viruses containing either wild-type or temperature-sensitive (ts) A mutants of SV40. Cells transformed with the SV40 tsA chimera (HP-A1 and HP-A2), but not the SV40 wild-type chimera (HP-W1), were temperature sensitive for transformation. All three cell lines expressed trophoblast-specific genes, including PSG and the alpha- and beta-subunits of hCG. Human CG alpha expression was greatly stimulated by (Bu)2cAMP in all three cell lines; shifting HP-A1 and HP-A2 cells to the nonpermissive temperature (39.5 C) further increased hCG alpha expression. At both 33 C (permissive temperature) and 39.5 C, the transformed placental cells expressed PSG mRNAs of 2.2 and 1.7 kilobases; expression was greatly stimulated by sodium butyrate. In the absence of an inducer, the three placental lines synthesized a PSG of 64 kilodaltons (kDa). In the presence of butyrate, they synthesized PSGs of 72, 64, and 54 kDa, similar to the placental PSGs. However, in placenta the predominant species is the 72-kDa product. At 39.5 C, butyrate selectively increased synthesis of the 72-kDa PSG in HP-A1 and HP-A2 cells. To characterize PSG promoter activity, we constructed chloramphenicol acetyltransferase (CAT) fusion genes containing -809 to -44 basepairs up-stream of the translational start site of the PSG6 gene. Using transient expression assays, we demonstrated that the -809/-44 region of the PSG6 gene contained cis-acting sequences that can direct CAT expression in human placental cells. Sodium butyrate, which stimulates PSG expression, greatly increased CAT activity, indicating that butyrate-induced PSG expression is regulated primarily at the level of gene transcription.

Two new mutations in the glucose-6-phosphatase gene cause glycogen storage disease in Hungarian patients.

Glycogen storage disease type 1a (von Gierke disease, GSD-1A) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the identification of the mutations causing GSD-1a. This, in turn, allows the development of non-invasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. Here we report on two new mutations E110Q and D38V causing GSD-1a in two Hungarian patients. The analyses of these mutations by site-directed mutagenesis followed by transient expression assays demonstrated that E110Q retains 17% of G6Pase enzymatic activity while the D38V abolishes the enzymatic activity. The patient with the E110Q has G222R as his other mutation. G222R was also shown to preserve about 4% of the G6Pase enzymatic activity. Nevertheless, the patient presented with the classical severe symptomatology of the GSD-1a.

Glycogen storage disease type 1a in Israel: biochemical, clinical, and mutational studies.

Glycogen storage disease type 1a (von Gierke disease, GSD 1a) is caused by the deficiency of microsomal glucose-6-phosphatase (G6Pase) activity which catalyzes the final common step of glycogenolysis and gluconeogenesis. The recent cloning of the G6Pase cDNA and characterization of the human G6Pase gene enabled the characterization of the mutations causing GSD 1a. This, in turn, allows the introduction of a noninvasive DNA-based diagnosis that provides reliable carrier testing and prenatal diagnosis. In this study, we report the biochemical and clinical characteristics as well as mutational analyses of 12 Israeli GSD 1a patients of different families, who represent most GSD 1a patients in Israel. The mutations, G6Pase activity, and glycogen content of 7 of these patients were reported previously. The biochemical data and clinical findings of all patients were similar and compatible with those described in other reports. All 9 Jewish patients, as well as one Muslim Arab patient, presented the R83C mutation. Two Muslim Arab patients had the V166G mutation which was not found in other patients' populations. The V166G mutation, which was introduced into the G6Pase cDNA by site-directed mutagenesis following transient expression in COS-1 cells, was shown to cause complete inactivation of the G6Pase. The characterization of all GSD 1a mutations in the Israeli population lends itself to carrier testing in these families as well as to prenatal diagnosis, which was carried out in 2 families. Since all Ashkenzai Jewish patients harbor the same mutation, our study suggests that DNA-based diagnosis may be used as an initial diagnostic step in Ashkenazi Jews suspected of having GSD 1a, thereby avoiding liver biopsy.

Subtle differences in human pregnancy-specific glycoprotein gene promoters allow for differential expression.

Eleven pregnancy-specific glycoprotein (PSG) genes reside on human chromosome 19. The sequence of these genes is extremely similar and that similarity extends to their putative control regions. However, the expression pattern of each PSG gene differs in the placenta, the primary site of PSG synthesis. To understand the molecular mechanisms underlying differential PSG expression, we characterized promoter elements of six PSG genes. We have shown previously that nucleotides -172 to -34 with respect to the translation start site constitute a minimal promoter in the PSG12 gene (class 1). We now show that PSG1-I and PSG3 are also members of class 1 genes. In contrast, only nucleotides -172 to -80 are necessary for promoter activity in PSG5, PSG6, and PSG11 genes (class 2). Class 2 genes contain a perfect Sp1 recognition sequence (CCCCGCCC) at nucleotides -148 to -141 which is necessary for promoter activity. Placental cell extracts formed three protein-DNA complexes with nucleotides -172 to -80 of all six PSG genes. One of the components of these complexes is an Sp1-like molecule. We have previously reported activator sequences within nucleotides -83 to -34 in PSG12. We now show that a 50-kDa protein binds to this region of PSG12, and the resultant complex can be supershifted by a monoclonal antibody to PEA3.

Structural analysis of the locus containing the human C-reactive protein gene and its related pseudogene.

The gene for human C-reactive protein (CRP) is mapped within a 34-kilobase pair genomic DNA segment identified by chromosome walking through overlapping DNA fragments cloned into a lambda phage library. Within 16 kilobase pairs upstream and downstream of the locus for the authentic CRP gene, only one other sequence homologous to that for CRP could be found. Sequencing analysis indicates this sequence to be a pseudogene with 50-80% region-specific homology. Comparison of the authentic CRP gene cloned from genomic DNA libraries independently prepared from three patients indicates no difference in the 5' and 3' flanking region, promoter region, or coding sequence. Only a polymorphism in the length of the poly(GT) stretch located in the intron is observed. There appears to be only one gene locus and copy per haploid chromosome for the authentic CRP gene and its pseudogene.

Asparagine-linked oligosaccharides are localized to a luminal hydrophilic loop in human glucose-6-phosphatase.

Deficiency of glucose-6-phosphatase (G6Pase), an endoplasmic reticulum transmembrane glycoprotein, causes glycogen storage disease type 1a. We have recently shown that human G6Pase contains an odd number of transmembrane segments, supporting a nine-transmembrane helical model for this enzyme. Sequence analysis predicts the presence of three potential asparagine (N)-linked glycosylation sites, N96TS, N203AS, and N276SS, conserved among mammalian G6Pases. According to this model, Asn96, located in a 37-residue luminal loop, is a potential acceptor for oligosaccharides, whereas Asn203 and Asn276, located in a 12-residue cytoplasmic loop and helix 7, respectively, would not be utilized for this purpose. We therefore characterized mutant G6Pases lacking one, two, or all three potential N-linked glycosylation sites. Western blot and in vitro translation studies showed that G6Pase is glycosylated only at Asn96, further validating the nine-transmembrane topology model. Substituting Asn96 with an Ala (N96A) moderately reduced enzymatic activity and had no effect on G6Pase synthesis or degradation, suggesting that oligosaccharide chains do not play a major role in protecting the enzyme from proteolytic degradation. In contrast, mutation of Asn276 to an Ala (N276A) destabilized the enzyme and markedly reduced enzymatic activity. We present additional evidence suggesting that the integrity of transmembrane helices is essential for G6Pase stability and catalytic activity.

Preparations and properties of crystalline porcine and bovine [Trp]B1 insulins.

[Trp]B1 analogs to porcine and bovine insulins have been obtained through selective reaction with Msc-ONSu, and followed by Edman degradation, condensation with Msc-Trp-ONp and deprotection. Through cellulose acetate electrophoresis, amino acid composition analysis, UV absorption spectrum and N-terminal analysis, it has been proved that the products are of purified [Trp]B1 insulin. Mouse convulsion assay shows that the biological activities of porcine and bovine [Trp]B1 insulins are 18 i.u./mg and 17 i.u./mg respectively, corresponding to about 70% of the original activity of native porcine insulin. Their structural details are under investigation in our laboratory.

Effects of sodium butyrate on the synthesis of human pregnancy-specific beta 1-glycoprotein.

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a polymorphic placental protein which shows strong sequence similarity with the oncofetal protein, carcinoembryonic antigen. To better understand the role of PS beta G in pregnancy, we examined its synthesis and regulation in placental fibroblasts, which had been shown to express the PS beta G gene. The major placental PS beta G is a 72-kDa glycoprotein, while the major fibroblast PS beta G is a 62-kDa species. Administration of sodium butyrate to these fibroblasts slightly stimulated the synthesis of the 62-kDa species but markedly increased the production of two additional PS beta Gs of 72 and 48 kDa. The similarity between the PS beta Gs synthesized by butyrate-treated fibroblasts and human placenta was confirmed by cell-free protein synthesis. Poly(A)+ RNA from butyrate-treated fibroblasts and placenta directed the synthesis of two polypeptides of 48 and 36 kDa, which form the polypeptide backbone of the 72- and 48-kDa glycoproteins. Moreover, the predicted molecular weights of PS beta Gs encoded by the two types of PS beta G cDNA clones were 48,000 and 36,000. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity at the 5' region (designated PSG-5') but differ in sequences at their 3' regions. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases from placental fibroblasts. Butyrate increased the steady-state levels of all three mRNAs. Ribonuclease protection analysis showed that butyrate increased the PS beta G mRNAs containing the PSG-5' or PSG93-specific sequence to approximately 20% of human placental levels. However, unlike human term placenta, which predominantly expressed PS beta G mRNAs with 3'-sequences similar to PSG16/PSG93, the butyrate-treated fibroblasts expressed roughly equal levels of PS beta G mRNAs with the PSG16/PSG93-3' and PSG95-3' ends. All PS beta G cDNAs identified encode proteins with distinct carboxyl termini, suggesting that the composition of the 72-kDa species in placenta and butyrate-treated fibroblasts is likely to be different. Placental fibroblasts provide a unique model for the study of the mechanisms responsible for the differential expression of the PS beta G gene.

Cloning and expression of genes encoding human pregnancy-specific glycoproteins.

The pregnancy-specific glycoproteins (PSGs) of the human placenta and the carcinoembryonic antigens comprise a subfamily within the immunoglobulin superfamily. There may be as many as 20 different PSG genes which are predominantly expressed in the placenta. As an initial step toward understanding the control of PSG expression, we isolated and characterized two nearly identical PSG genes, PSG1 and PSG1-I. PSG1, which lacks exon 1 (5'/L), but contains exons 2 (L/N), 3 (A1), 4 (A2), and 5 (B2-C), encodes five previously identified type I transcripts, PSG1a, 1b, 1c, 1d, and 1e in a L/N-A1-A2-B2-C domain arrangement. PSG1-I, which contains a complete transcriptional unit consisting of exons 5'/L, L/N, A1, and B2-C, encodes type II PSG transcripts in a L/N-A1-B2-C domain arrangement. The predicted PSG1-I-encoded proteins share nearly complete sequence identity with the PSG1-encoded members, except the latter contain extra A domains. Amplification by polymerase chain reaction of placental or hydatidiform mole cDNA demonstrates that PSG1-I is a functional type II PSG gene. Using transient expression assays, we demonstrated that the -834/-34 region upstream of the translational start site of the PSG1-I gene contained the PSG promoter elements and the -834 to -456 region contained negative control elements. Sodium butyrate, an inducer of PSG synthesis, greatly stimulated expression of all PSG1-I-chloramphenicol acetyltransferase (CAT) fusion gene constructs. However, butyrate was at least 2-fold more effective in stimulating CAT activity of fusion genes containing upstream sequences (-834 to -576) than those containing proximal sequences (-456 to -172), suggesting two regions in the PSG1-I gene that mediate the butyrate response.

Purification and N-terminal partial sequence of anti-epilepsy peptide from venom of the scorpion Buthus martensii Karsch.

An anti-epilepsy peptide (AEP) was isolated and purified from venom of the scorpion Buthus martensii Karsch. The purification procedure included CM-Sephadex C-50 chromatography, gel filtration on Sephadex G-50 and DEAE-Sephadex A-50 chromatography. Its homogeneity was demonstrated by pH 4.3 polyacrylamide-disc-gel electrophoresis, focusing electrophoresis and SDS/polyacrylamide-disc-gel electrophoresis. The Mr of this peptide, calculated from measurements in SDS/15%-polyacrylamide-disc-gel and SDS/20%-polyacrylamide-disc-gel electrophoresis, is 8300. The isoelectric point is 8.52 by pH 8-9.5-range isoelectric focusing. No haemorrhagic or toxic activities were found. No toxicity was found even after the dose reached 28 mg/kg. The pharmacological tests showed that the AEP had no effect on heart rate, blood pressure or electrocardiogram, but strongly inhibited epilepsy induced by coriaria lactone and cephaloridine. The fluorescence spectrum showed that the peptide has a strong emission peak at 337 nm. Amino acid analysis suggested that the AEP is composed of 66 residues from 18 amino acids and has an Mr of 8290. The sequence of the first 50 N-terminal residues is as follows: Asp-Gly-Tyr-Ile-Arg-Gly-Ser-Asp-Asn-Cys-Lys-Val-Ser-Cys-Leu-Leu-Gly-Asn- Glu-Gly - Cys-Asn-Lys-Glu-Cys-Arg-Ala-Tyr-Gly-Ala-Ser-Tyr-Gly-Tyr-Cys-Trp-Thr-Val- Lys-Leu - Ala-Gln-Asp-Cys-Glu-Gly-Leu-Pro-Asp-Thr-.

Transmembrane topology of glucose-6-phosphatase.

Deficiency of microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis, causes glycogen storage disease type 1a, an autosomal recessive disorder. Characterization of the transmembrane topology of G6Pase should facilitate the identification of amino acid residues contributing to the active site and broaden our understanding of the effects of mutations that cause glycogen storage disease type 1a. Using N- and C-terminal tagged G6Pase, we show that in intact microsomes, the N terminus is resistant to protease digestion, whereas the C terminus is sensitive to such treatment. Our results demonstrate that G6Pase possesses an odd number of transmembrane helices, with its N and C termini facing the endoplasmic reticulum lumen and the cytoplasm, respectively. During catalysis, a phosphoryl-enzyme intermediate is formed, and the phosphoryl acceptor in G6Pase is a His residue. Sequence alignment suggests that mammalian G6Pases, lipid phosphatases, acid phosphatases, and a vanadium-containing chloroperoxidase (whose tertiary structure is known) share a conserved phosphatase motif. Active-site alignment of the vanadium-containing chloroperoxidase and G6Pases predicts that Arg-83, His-119, and His-176 in G6Pase contribute to the active site and that His-176 is the residue that covalently binds the phosphoryl moiety during catalysis. This alignment also predicts that Arg-83, His-119, and His-176 reside on the same side of the endoplasmic reticulum membrane, which is supported by the recently predicted nine-transmembrane helical model for G6Pase. We have previously shown that Arg-83 is involved in positioning the phosphate during catalysis and that His-119 is essential for G6Pase activity. Here we demonstrate that substitution of His-176 with structurally similar or dissimilar amino acids inactivates the enzyme, suggesting that His-176 could be the phosphoryl acceptor in G6Pase during catalysis.

Ontogeny of the murine glucose-6-phosphatase system.

A deficiency in microsomal glucose-6-phosphatase (G6Pase) activity causes glycogen storage disease type 1 (GSD-1), a clinically and biochemically heterogeneous group of diseases. It has been suggested that catalysis by G6Pase involves multiple components, with defects in the G6Pase catalytic unit causing GSD-1a and defects in the putative substrate and product translocases causing GSD-1b, 1c, and 1d. However, this model is open to debate. To elucidate the G6Pase system, we have examined G6Pase mRNA expression, G6Pase activity, and glucose 6-phosphate (G6P) transport activity in the murine liver and kidney during normal development. In the liver, G6Pase mRNA and enzymatic activity were detected at 18 days gestation and increased markedly at parturition, before leveling off to adult levels. In the kidney, G6Pase mRNA and enzymatic activity appeared at 19 days gestation and peaked at weaning, suggesting that kidney G6Pase may have a different metabolic role. In situ hybridization analysis demonstrated that, in addition to the liver and kidney, the intestine expressed G6Pase. Despite the expression of G6Pase in the embryonic liver, microsomal G6P transport activity was not detectable until birth, peaking at about age 4 weeks. Our study strongly supports the multicomponent model for the G6Pase system.