Characterization of a phosphoglycerate kinase deficiency variants not associated with hemolytic anemia.

The properties of a variant phosphoglycerate kinase (PGK) found in a large German clan were examined. The normal and variant enzymes, isolated by affinity chromatography, have the same molecular weight, specific activity, substrate affinity, and nearly identical pH-optima. Using immunoinactivation and immunodiffusion, the same specific activity for both forms was again determined. Since the enzymatic activity in older and younger erythrocytes varied only slightly, and since the specific activity of the variant was normal, the variant seems to be stable in vivo. This suggests that the decreased enzyme content is due to a decreased synthesis rate. The variant PGK described here is distinctly different from the known PGK variants and has been designated as "PGK München."     

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.     

A case of congenital non-spherocytic hemolytic anemia caused by enzyme deficiency in pyruvate kinase

About a familial observation of PK deficiency, the authors emphasize the important clinical and biochemical heterogeneity. Interest of isotopic explorations in the therapeutic decision of splenectomy.     

Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

Properties of thiamin triphosphate-synthesizing activity of chicken cytosolic adenylate kinase and the effect of adenine nucleotides

Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme.     

Bordetella pertussis adenylate cyclase toxin. Structural and functional independence of the catalytic and hemolytic activities.

The Bordetella pertussis calmodulin-dependent adenylate cyclase (CyaA) is a 1706-residue-long toxin, endowed with hemolytic activity. We have constructed B. pertussis mutant strains producing modified CyaAs devoid of adenylate cyclase activity. Our results show that such modified CyaAs display hemolytic activity identical to the wild-type toxin, thus demonstrating that the hemolytic activity is independent of the adenylate cyclase activity. Furthermore, B. pertussis and Escherichia coli strains producing CyaA lacking the catalytic domain (residues 1-373) were constructed. The truncated protein exhibits hemolytic activity comparable to the wild-type toxin, thus establishing that the carboxyl-terminal 1332 residues alone are endowed with hemolytic activity. Together, these findings show that adenylate cyclase and hemolytic activities are located in two distinct regions of the molecule (respectively, approximately amino acids 1-400 and 401-1706) and that the two regions of CyaA are functionally independent.     

Human glycerol kinase deficiency with hyperglycerolemia and glyceroluria.

Two brothers with a previously unidentified syndrome of severe osteoporosis and neuromuscular disease have elevated concentrations of glycerol in serum and urine. A deficiency of glycerol kinase in leukocytes of both patients is described.     

A variant glucose-6-phosphate dehydrogenase Gd(-) Chiapas associated with moderate enzyme deficiency and occasional hemolytic anemia

Summary Erythrocyte glucose-6-phosphate deficiency is an X-chromosomal-linked hereditary trait often associated with hemolytic anemia. This report defines a new variant designated as Gd(-) Chiapas, which was found in a subject with occasional hemolytic jaundice. The red cell enzyme activity of the subject is about 15% of normal. The variant enzyme is thermolabile in vitro and has faster-than-normal anodal electrophoretic mobility and stronger-than-normal substrate affinity. The patient's hemolytic problem might be correlated with instability of the variant enzyme under physiologic stress.This study was supported in part by the US Public Health Service Grant HL-15125.     

A single amino acid substitution enhances the catalytic activity of family 11 xylanase at alkaline pH

Random mutagenesis of the gene encoding family 11 xylanase was used to obtain alkalophilic mutants. The catalytic domain of the chimeric enzyme Stx15, which was constructed from Streptomyces lividans xylanase B and Thermobifida fusca xylanase A, was mutated using error-prone PCR and screened for halo formation on dye-linked xylan plates and activity toward soluble xylan. A positive mutant, M1011, was isolated, and it was found that mutation A49V was responsible for the alkalophilicity of the mutant. Mutation A49V increased the specific activity at pH 9.1 and the stability of mutant A49V was not significantly different from that of Stx15 at 60 degrees C. Both enzymes retained more than 90% of their relative activity from pH 4.7 to 9.1 after 1 h of incubation at 60 degrees C. Analysis of the kinetic parameters at various pH values showed that the A49V mutation reduced the Km in the alkaline pH range, resulting in the higher specific activity of the A49V mutant enzyme.     

Thyroid-stimulating hormone (TSH) deficiency caused by a single base substitution in the CAGYC region of the beta-subunit.

Congenital isolated thyroid-stimulating hormone (TSH) deficiency is an autosomal recessive disease that manifests as hypothyroidism (cretinism), causing severe mental and growth retardations. Patients were found to have a single base substitution in the codon for the 29th amino acid of the TSH beta subunit gene. The alteration is in the center of the so-called CAGYC region, which consists of an amino acid sequence conserved among all of the known glycoprotein hormone beta subunits. No other nucleotide substitutions have been found in the gene thus far sequenced. Microinjection of the mutated beta mRNAs into Xenopus laevis oocytes led to the formation of conformationally altered beta polypeptides that could not associate with alpha subunits. The mutation created a new recognition site for the enzyme MaeI. Southern blot hybridization of genomic DNA digested with MaeI showed that the patients were homozygous and their parents were heterozygous for the mutation. This test was also used to examine other family members for the disease.     

A case of congenital non-spherocytic hemolytic anemia caused by triose phosphate isomerase deficiency. Prenatal diagnosis

Prenatal diagnosis: The authors present a personal case of triose-phosphate-isomerase deficiency. Clinically the deficiency associates a constitutional non spherocytic anemia, paroxystic and precocius, and neuromuscular symptoms (axial hypotonia and limb palsies). A diaphragmatic paralysis may complicate the syndrome. Infections are frequent. Survival rarely goes beyond 5 years of age. Biochemical exams show the ubiquity of the deficiency. The physiopathology remains obscure. The TPI deficiency is heritable (autosomal recessive transmission). The gene has been mapped on the short arm of the chromosome 12. Prenatal diagnosis is possible.     

Hemolytic anemia due to pyruvate kinase deficiency: characterization of the enzymatic activity from eight patients.

We have studied the red cell pyruvate kinase (PK) variants from eight patients representing five families with pyruvate kinase deficiency-associated hemolytic anemia. The kinetic properties, electrophoretic mobilities, and immunological reactivity with anti-normal red cell pyruvate kinase were determined. The patients differ in the severity of their clinical condition and in the molecular properties of their red cell pyruvate kinase variants. The most seriously affected patient (PK Beaverton) has no electrophoretically demonstrable red cell isozymes. The activity present is due to the M2 isozyme, however red cell isozyme can be detected immunologically. PK Molalla and PK Lake Oswego are thermolabile variants with normal kinetic parameters. PK Molalla, in addition, has altered electrophoretic mobility. PK Multnomah and PK Milwaukie have decreased affinity for the substrate phosphoenolpyruvate, and PK Multnomah also has altered electrophoretic mobility. PK Coos Bay shows electrophoretic variation and a slightly decreased affinity for phosphoenolpyruvate consistent with an increased modulating effect of fructose-1,6-diphosphate.