Tissue variability of the phosphotransferases adenylate kinase (EC: 2.7.4.3.) and pyruvate kinase (EC: 2.7.1.40.)

Summary Studies of the tissue variability of human AK and PK are reported. Homozygous probands of phenotype AK 1, show five electrophoretic positions with AK activity in different tissues and six positions with PK activity. In one of the 20 probands studied the brain, not however any other tissue, revealed a different zymogram. It can be followed therefore that different isozymes can be present in different tissues.Head: Prof. Dr. Dr. H. BaitschSupported by the Deutsche Forschungsgemeinschaft.     

Phosphoglucomutase (EC 2.7.5.1.) and adenylate kinase (EC 2.7.4.3.) typings in Koreans and Irish

Summary PGM₁ and AK phenotypes were determined in samples from Korea and Ireland. the frequencies of PGM ₁ ¹ genes amount to 0.916 in Koreans and 0.864 in Irish. AK¹ frequencies come to 0.933 in Koreans and 0.873 in Irish.Supported by the Deutsche Forschungsgemeinschaft.     

Ontogenesis and independent genetic control of the rat adenylate kinase isozymes (EC 2.7.4.3.)

The level of activity of cytoplasmic isozyme II of adenylate kinase (EC 2.7.4.3) appears to be genetically controlled in the rat (Rattus norvegicus). Adult animals of the Okamoto inbred strain exhibit a ninefold higher erythrocytic activity than the rats of the dilute agouti inbred strain. This difference seems to be due to two codominant autosomal alleles at a same locus. The study of the ontogeny of that enzyme in muscle in the two strains shows that the wellknown postnatal activity increase of that isozyme is reduced in the low activity (dilute agouti) strain. However, the mitochondrial isozyme III activity is not similarly regulated as no difference between the two strains has been observed.     

Glutathione reductase (EC 1.6.4.2.) in experimental trypanosomiasis

The activity of glutathione reductase (GHSR) in extracts of kidney, liver and testis of rats infected with Trypanosoma congolense decreased with every wave of parasitemia. The implications of these observations as they relate to the risk of oxidative stress are discussed.     

The association of yeast phosphoglycerate kinase (EC 2.7.2.3).

1. A mol.wt. of 40030 +/- 830 has been estimated for phosphoglycerate kinase in concentrations less than 0.1 g/100 cm3 comparing favourably with expected values from X-ray diffraction measurements by 10% lower than the previously reported molecular weights made at higher concentrations. 2. The so20w, was estimated to be 3.12(+/-0.02)x10(-13)s and the coefficient had a low concentration dependency giving a g value (concentration-dependency) of 2.3 +/- 1.6cm3 .g-1. This agrees with previous qualitative observations. 3. By using fluctuation-intensity spectroscopy, the D20,w was estimated to be 7.4(+/-0.2)x10(-11)m2.s-1, and this was indistinguishable from the D20,w calculated from ultracentrifuge results. The water of hydration was estimated to be 0.46 g/g of protein. 4. It is inferred from the estimates that phosphoglycerate kinase associates with an interaction coefficient at 20 degrees C for monomer/dimer of between 10 and 12 cm3.g-1. 5. The ratio of molecular asymmetry (a/b) was estimated to be 2.5+/-0.2 from the values of D20,w and water of hydration. This compares favourably with the ratio from the overall dimensions estimated from X-ray diffraction measurements.     

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.     

Zur Formalgenetik der Phosphoglucoseisomerase (EC: 5.3.1.9)

Zusammenfassung Es werden Befunde aus einer Sippe mit PGI-Defizienz mitgeteilt. Die beiden Patienten haben eine nichtsphärocytäre hämolytische Anämie, sie besitzen den homozygoten Phänotypus PGI9. Die Eltern sind heterozygot PGI 9-1. Der Zymogrammvergleich von Eltern und Kindern spricht für die Hypothese, daß die PGI ein dimeres Molekül sei, das normalerweise aus identischen Polypeptidketten aufgebaut ist.

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Formal genetics of phosphoglucoseisomerase investigations of a family with PGI-Deficiency

Summary In a family two children exhibited a non-sphaerocytic hemolytic anemia; they revealed the homozygous phenotype PGI9. The parents are heterozygous PGI 9-1. The comparison of the zymogram pattern of both parents and children allows the conclusion that the PGI molecule is a dimer which has identical subunits in homozygous individuals.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Establishment of thymidine kinase (EC 2.7.1.21)-deficient mutants of human monocytic cell line U-937

Using morphologic, enzyme-cytochemical, and immunocytochemical methods, the functional diversity of the mononuclear phagocyte system can be studied only to a limited extent. Therefore, enzyme-deficient monocyte/macrophage cell lines have been established as technical prerequisites for the generation of monocyte/macrophage hybrids by applying selective media. After mutation with ethylmethanesulfonate, six clones of U-937 were selected against increasing concentrations of 5'-bromodesoxyuridine; these clones are defective in thymidine kinase (EC 2.7.1.21), as shown by autoradiography and direct measurement of [3H]thymidine uptake. A broad marker panel indicates that the clones could be appropriate for the establishment of human monocyte/macrophage hybrids.     

Zur populationsgenetik der phosphoglucoseisomerase (EC: 5.3.1.9)

Summary In a population sample of 728 unrelated individuals from Marburg one variant PGI 5-1 was found.

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Direktor: Prof. Dr. Dr. H. Ritter

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Direktor: Prof. Dr. G. G. Wendt

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Amblyomma americanum (L.): protein kinase C-independent fluid secretion by isolated salivary glands.

Protein kinase C activity was partially purified from tick salivary glands by fast protein liquid chromatography anion-exchange chromatography. Enzyme activity was stimulated by Ca2+, phosphatidylserine, and diacylglycerol with the highest activity observed in the presence of all three modulators. Enzyme activity was inhibited by a synthetic pseudosubstrate peptide with an amino acid sequence resembling the protein kinase C substrate phosphorylation site. The protein kinase C activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), when added to whole in vitro salivary glands previously prelabeled with 32P, stimulated the phosphorylation of salivary gland proteins. Activators of protein kinase C (phorbol ester or OAG) did not stimulate fluid secretion by isolated tick salivary glands. OAG and phorbol ester had only minimal affects on the ability of dopamine to stimulate secretion by isolated salivary glands and dopamine's ability to increase salivary gland cyclic AMP.     

Red-cell galactose-1-phosphate-uridyl transferase (EC: 2.7.7.12)

Summary For this study 94 families with 195 children were investigated. The segregation of the children's phenotypes is in agreement with the formal two-allele model. Close linkage has been ruled out for a number of informative markers.

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Zusammenfassung Der Polymorphismus der Galaktose-1-Phosphat-Uridyl-Transferase wurde an 94 Familien mit 195 Kindern untersucht. Die Aufspaltung der Kinderphänotypen entspricht dem formalgenetischen Modell. Für eine Reihe informativer Systeme wurde enge Kopplung ausgeschlossen.

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Supported by the Deutsche Forschungsgemeinschaft.