The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase

The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase, comprising 239 residues, was determined. The sequence was deduced from the four cyanogen bromide fragments, and from the peptides derived from these fragments after digestion with a number of proteolytic enzymes. Comparison of this sequence with that of the yeast glycolytic enzyme, phosphoglycerate mutase, shows that these enzymes are 47% identical. Most, but not all, of the residues implicated as being important for the activity of the glycolytic mutase are conserved in the erythrocyte diphosphoglycerate mutase.     

The isolation and partial characterization of diphosphoglycerate mutase from human erythrocytes.

Diphosphoglycerate mutase has been purified to homogeneity from outdated human erythrocytes. The native enzyme has a molecular weight of 57 000 as determined by equilibrium centrifugation and exclusion chromatography. Disc gel electrophoresis in the presence of sodium dodecyl sulfate yields a single protein band with a molecular weight of about 26 500, indicating that diphosphoglycerate mutase is comprised of two subunits of similar mass. The enzyme exhibits the following intrinsic activities: diphosphoglyceratemutase, monophosphoglycerate mutase, and 2,3-diphosphoglycerate phosphatase. The latter activity is enhanced in the presence of either organic or inorganic anions. Glycolate-2-P, particularly, has a profound activating effect. Nonspecific phosphatase and enolase activities are absent. The enzyme has an extinction coefficient at 280 nm of 1.65 cm2/mg. The amino acid composition of the homogeneous protein has been determined.     

Effects of salts and pH on the rate of erythrocyte diphosphoglycerate mutase

Erythrocyte diphosphoglycerate mutase is inhibited by several inorganic salts, the extent of the effect being characteristic of the anionic component, i.e., at ionic strength of about 0.1, SO₄^2? > Cl^? > CH₃COO^?. Using a partially purified enzyme preparation from human red blood cells, kinetic constants were determined in the presence of 0.1 m KCl to simulate the ionic environment of the cell. At pH 7.5, the addition of salt caused a 10-fold increase in the K_m of 1,3-diphosphoglycerate and a 46-fold increase in the K_i of 2,3-diphosphoglycerate. There was no effect of salt on the K_m of 3-phosphoglycerate or on the maximal velocity of the reaction. In the presence of 0.1 m KCl, the _i of inorganic phosphate increased from 0.3 mm to 0.6 mm. The K_m of 1,3-diphosphoglycerate was pH dependent, the values obtained being 3.6 μm at pH 6.75, 3.1 μm at pH 7.24, and 6.7 μm at pH 7.75. The K_i values for 2,3-diphosphoglycerate under the same conditions were: 12 μm at pH 6.75, 20μm at pH 7.24, and 53 μm at pH 7.75. The relative maximal velocity of the reaction has been evaluated over the same pH range. The maximal activity of the enzyme measured at 25 °C and pH 7.5 was 2 units/min/ml of packed red cells. From these studies, it is concluded that the effective enzymatic rate increases fourfold when the pH increases from 6.75 to 7.75.     

Hepatic phosphorylase deficiency: a biochemical study

Two boys with hepatomegaly had increased glycogen content in the liver and no activity of liver phosphorylase, even in the presence of 5′ AMP. The biochemical differences between phosphorylase- and phosphorylase $\text{b}$ kinase deficiency are discussed, and a differential diagnostic procedure proposed.     

Location of phosphoglycerate mutase in rat skeletal muscle. An immunocytochemical and biochemical study

The subcellular distribution of phosphoglycerate mutase was studied by immunogold techniques. With the aid of highly affinity-purified anti-phosphoglycerate mutase antibodies, the enzyme was found in both cytosol and nucleus of rat skeletal muscle. No evidence of interaction with contractile proteins was observed in cytosol. Nuclear location was also confirmed biochemically using purified nuclear preparations from rat skeletal muscle. Only one immunoreactive nuclear band was observed by Western blot experiments and corresponded to that of phosphoglycerate mutase mobility. Activity measurements from nuclear extracts showed that 25% of total specific activity is found in the nuclei.     

Polyanionic inhibitors of phosphoglycerate mutase: combined structural and biochemical analysis.

The effects that the inhibitors inositol hexakisphosphate and benzene tri-, tetra- and hexacarboxylates have on the phosphoglycerate mutases from Saccharomyces cerevisiae and Schizosaccharomyces pombe have been determined. Their Kivalues have been calculated, and the ability of the inhibitors to protect the enzymes against limited proteolysis investigated. These biochemical data have been placed in a structural context by the solution of the crystal structures of S. cerevisiae phosphoglycerate mutase soaked with inositol hexakisphosphate or benzene hexacarboxylate. These large polyanionic compounds bind to the enzyme so as to block the entrance to the active-site cleft. They form multiple interactions with the enzyme, consistent with their low Kivalues, and afford good protection against limited proteolysis of the C-terminal region by thermolysin. The inositol compound is more efficacious because of its greater number of negative charges. The S. pombe phosphoglycerate mutase that is inherently lacking a comparable C-terminal region has higher Kivalues for the compounds tested. Moreover, the S. pombe enzyme is less sensititive to proteolysis, and the presence or absence of the inhibitor molecules has little effect on susceptibility to proteolysis.     

Cutting edge: TRAIL deficiency accelerates hematological malignancies

TNF apoptosis-inducing ligand is attracting considerable interest as a potential extrinsic tumor suppressor mechanism, although previous reports have conveyed somewhat contrasting views regarding the likely importance of this pathway. In this study, we provide the first evaluation of spontaneous tumor formation over the life span of TRAIL-deficient mice. Interestingly, >25% of these mice do develop lymphoid malignancies after 500 days of life. TRAIL suppressed the initiation and development of both tumors of lymphoid and stromal origin in the context of the loss of at least one p53 allele. Specific examination of the role of TRAIL in Her2/neu oncogene-driven mammary epithelial cancer revealed no critical role for TRAIL despite the inherent TRAIL sensitivity of such mammary carcinomas. Overall, the data indicate an important function of TRAIL in controlling carcinogenesis, but suggest that further examination of this pathway in epithelial malignancies is warranted.     

Prolidase deficiency: biochemical classification of alleles

Prolidase (E.C.3.4.13.9) is a homodimeric enzyme encoded at a locus on chromosome 19. Prolidase deficiency is an autosomal recessive disorder with a highly variable clinical phenotype. We purified prolidase to homogeneity from normal human fibroblasts, raised a monospecific rabbit antiserum, and studied biosynthesis of the subunit in normal and prolidase--deficient fibroblasts. Pulse-chase immunoprecipitation experiments showed that the subunit is synthesized and retained in cytosol as a 58-KDa polypeptide. Three types of mutations were identified in six prolidase-deficient cell strains; half conferred a CRM-negative phenotype, while the CRM-positive mutations were of two types, one mutation encoding an enlarged subunit (60 KDa) and the others associated with subunits of normal size. Complementation analysis indicated that these mutations map to one locus. Normal subjects and obligate heterozygotes expressing CRM-negative mutations had thermostable prolidase activity at 50 degrees C in cell extracts, whereas heterozygotes expressing CRM-positive mutations had thermolabile activity under the same condition, implying negative allelic complementation in the putative heterodimer. The occurrence of prolidase-like activity about 5% of normal in amount but with a preference for substrate different from normal, in cells homozygous (or compound) for CRM-negative mutations, identified an alternative cleavage activity not encoded at the prolidase locus. Allelic heterogeneity at the major locus and the amount of alternative peptidase activity encoded elsewhere appear to be determinants of the associated and heterogeneous clinical phenotype.     

Familial thrombosis: inherited deficiency of antithrombin III.

Several members of a family living on the west coast of Scotland and on one of the islands off the coast had serious thrombotic disease. The plasma antithrombin III (ATIII) concentrations were measured by both functional and immunological assay in all available members of the family. Concentrations were 25% to 66% of normal in 12 people, including all seven with thrombotic disease. The inheritance pattern was characteristic of an autosomal dominant disorder. Thrombotic disease generally affected the leg, mesenteric, and axillary veins, although one man who had died before the study began had had severe arterial atheroma. In women the first thrombotic symptoms usually occurred during pregnancy. None of these patients have developed thrombotic symptoms until they were at least 18, so four younger members of the family who have ATIII deficiency but no thrombotic disease may eventually develop symptoms.