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Biomedical subjects

W K Krietsch

Publications and source records attributed to W K Krietsch.

At least 19 recordsLinked to original sources
PubMed1991

Triosephosphate isomerase deficiency: haemolytic anaemia, myopathy with altered mitochondria and mental retardation due to a new variant with accelerated enzyme catabolism and diminished specific activity.

A new triosephosphate isomerase (TPI) variant is described in an 8-year-old Turkish girl suffering from chronic haemolytic anaemia, myopathy and developmental retardation since early infancy. The enzyme activity profile revealed a generalized deficiency in erythrocytes, granulocytes, mononuclear blood cells, skeletal muscle tissue and cerebrospinal fluid. The concentration of enzyme substrate dihydroxyacetone phosphate was distinctly elevated. Biochemical examination showed accelerated enzyme deamidation, the first step in the normal catabolism of TPI during aging of the erythrocyte. The specific activity of the variant TPI, determined by antibody titration, was reduced to 61% of normal. Its heat stability was markedly decreased. Muscle biopsy and neuropsychological testing further clarified the pathogenesis of the disorder. A prevalent alteration of mitochondria similar to that seen in mitochondrial myopathy and an elevated amount of intracellular glycogen were found. The patient's retarded intellectual development was mainly due to impaired visual perception and sensory-motor co-ordination in addition to a lack of syllogistic reasoning. The findings indicate that the low TPI activity leads to a metabolic block of the glycolytic pathway and hence to a generalized impairment of cellular energy supply.

Anemia, Hemolytic↗
PubMed1987

On the interaction of bovine seminal RNase with actin in vitro.

Ribonuclease from bovine seminal plasma (RNase BS) interacts with skeletal muscle actin in the following way: it binds to actin with an apparent binding constant of 9.2 X 10(4) M-1 in 0.1 M KCl, induces the polymerization of actin below the critical concentration in depolymerization buffer, accelerates the salt-induced polymerization of actin even at a molar ratio of RNase to actin lower than 1/100, and bundles F-actin filaments. In the bundles the molar ratio of RNase to actin is about 0.66. Actin inhibits the enzymatic activity of RNase BS. RNase A from bovine pancreas, which is structurally almost identical to the subunits of RNase BS as well as a monomeric form of RNase BS, do not cross-link actin filaments and have a much smaller effect on the polymerization of actin. We conclude that the dimeric structure of the RNase BS, which consists of two identical subunits cross-linked by interchain disulfide bridges, is probably responsible for the bundling activity and the accelerating effect on the polymerization of actin.

Actins↗
PubMed1987

Sequential expression of maternally inherited phosphoglycerate kinase-1 in the early mouse embryo.

Enzyme activities of X-linked phosphoglycerate kinase (PGK-1) and autosomal glucose phosphate isomerase (GPI-1) were determined in intact mouse blastocysts and isolated inner cell masses (ICMs). Blastocysts were recovered from the uterus on day 4 of gestation and cultured overnight in vitro. ICMs were isolated by treatment with calcium ionophore A23187. On day 4, approximately 35% of the total activity of both PGK-1 and GPI-1 was located in the ICM. After overnight culture, the PGK-1 activity of the whole blastocyst nearly doubled, due to the activation of only the maternally derived gene coding for PGK-1. In the ICM, however, a pronounced decrease of PGK-1 activity was measured: only 10% of the total PGK-1 activity was measured in the ICM on day 5. In contrast to PGK-1, GPI-1 activity of the intact blastocyst remained stable from day 4 to day 5. In the ICM, the GPI-1 activity did decline, but to a lesser extent than PGK-1 activity: 20% of total GPI-1 activity was found in the ICM on day 5. These results, when compared with the data of Handyside and Hunter, suggest that the decline in GPI-1 activity in the ICM is due to a change in the ratio of trophectoderm (TE) to ICM cells. The greater reduction of PGK-1 activity in the ICM cannot, however, be explained solely by this mechanism. To explain the observed additional decrease, we postulate that Pgk-1 is not activated in the ICM prior to day 6. This implies that on day 4 maternal Pgk-1 is activated in the TE exclusively.

Animals↗
PubMed1986

Expression of X-linked phosphoglycerate kinase in early mouse embryos homozygous at the Xce locus.

The expression of maternally derived X-chromosomal Pgk-1 alleles was investigated in oocytes and early embryos of mice carrying different alleles (Xcea, Xcec) of the X-chromosome controlling element (Xce) locus. Pgk-1 allelic expression was determined by measuring their gene products using Cellogel electrophoresis and a sensitive fluorimetric enzyme assay. In addition to the already existing mouse strain of the genotypes Pgk-1a Xcec and Pgk-1b Xcea, a new line was bred carrying the combination Pgk-1b Xcec. The X chromosomes carrying the combinations Pgk-1a Xcec and Pgk-1b Xcec were of feral origin, whereas Pgk-1b Xcea was derived from a laboratory line. Our results using Xcec homozygous females confirm that maternal Pgk-1 is already expressed on day 4 of embryogenesis, thus substantiating data previously obtained using Xcea/Xcec heterozygous females. This finding also demonstrates that the timing of reactivation of maternal Pgk-1 is not influenced by the Xce locus. Furthermore, we found that oocytes from Xcec homozygous females have a balanced PGK-1 A/PGK-1 B allozyme ratio (50:50), whereas in oocytes obtained from Xcea/Xcec heterozygotes, the PGK-1 allozyme ratio is about 60:40. In tissues of adult Xce homozygous females, the PGK-1 allozymes are also balanced, whereas in Xcea/Xcec heterozygous females, the ratio is about 35:65. In addition to the relative activity of the PGK-1 allozymes, we also measured the absolute activity of PGK-1 in oocytes obtained from three types of Xce homozygous females.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1985

Preferential expression of the maternally inherited X-linked phosphoglycerate kinase allele in human erythrocytes.

The stability of allelic gene expression of X-linked phosphoglycerate kinase was studied in seven carriers of a rare genetic variant named PGK München. The enzymatic activities in erythrocytes of five heterozygous females and three hemizygous males were determined repeatedly over a period of 10 years (1975-1984) and shown to remain constant. As the phosphoglycerate kinase activity is lower in cells expressing the PGK München allele, the ratio of the two cell types in all heterozygous females of the PGK München kindred could be calculated from the PGK activity and from the known allozyme activities in erythrocytes of homozygous wild type or hemizygous PGK München carriers. Since the maternal or paternal origin of both alleles is known from the pedigree, the quantitative expression of the maternally derived allozyme in heterozygous women could be determined. In heterozygous carriers the cell pool expressing the maternally inherited allele was significantly increased, independently, of the PGK allele linked to the maternal X chromosome (P less than 0.001). Our data show that inactivation of one of the two X chromosomes in human female erythropoietic stem cell precursors may be non-random, at least in the kindred and cell populations described here. The results are discussed in the context of random X chromosome inactivation (Lyon hypothesis).

Alleles↗
PubMed1985

Purification and properties of the phosphoglycerate mutase isozymes from the mouse.

The two homodimeric isozymes of phosphoglycerate mutase have been purified from murine kidney and muscle. No differences were observed in the Michaelis-Menten constant for the substrate 2-phospho-D-glycerate, in molecular weight, temperature and pH optima, when the purified isozymes were compared. The isozymes differ in their inhibition constants for phosphoenolpyruvate, in their Michaelis constants for 3-phospho-D-glycerate and 2,3-bisphospho-D-glycerate, their thermal and pH lability and in their sensitivity towards mercury ions.

Animals↗
PubMed1983

Comparison of the two purified allozymes (1B and 1A) of X-linked phosphoglycerate kinase in the mouse.

The two allelic isozymes (wild-type 1B and the electrophoretic variant 1A) of mouse X-linked phosphoglycerate kinase (PGK-1) have been purified by affinity chromatography. The following properties were determined for both forms: molecular weight, specific activity, nucleotide specificity, Km values of the four substrates, Ki of the ATP-ribosyladipoyldihydrazo-Mg complex, turnover number, activation energy, pH and ionic strength dependence, thermostability, content of free sulfhydryl groups, and antibody cross-reactivity. With the exception of specific activity and thermostability, both allozymes appear to be identical in all properties. The higher in vitro specific activity of the 1B allozyme may be due to the higher thermostability. No antigenic difference could be detected between the two allozymes.

Animals↗
PubMed1982

The expression of X-linked phosphoglycerate kinase in the early mouse embryo.

During early mouse embryogenesis, the activity of X-chromosomally linked maternal and paternal phosphoglycerate kinase (PGK-1) alleles was determined using electrophoretic separation of their gene products and a sensitive fluorometric enzyme assay. In the embryos collected from females homozygous for PGK-1b mated with PGK-1a males and vice versa, the paternally derived allozyme was first detected after implantation on day 6. Expression of the maternally inherited allele was studied in embryos from females heterozygous for PGK-1b and PGK-1a. From day 1 to day 4, the embryos maintained a constant ratio of enzyme activity of PGK-1B to PGK-1A. Prior to implantation of the embryos between day 4 and day 5, the activity ratio of the two PGK-1 allelic variants changed significantly due to the first appearance of newly synthesized PGK derived from the maternally inherited allele. Our data demonstrate a temporal difference in the onset of PGK synthesis depending on whether this particular gene product is of maternal or paternal origin. Therefore, we conclude that the maternal PGK-1 locus is already activated during late preimplantation development whereas the paternally inherited gene locus remains silent at the preimplantation stage but is subsequently expressed at approximately the time of X-chromosomal inactivation.

Animals↗
PubMed1980

The isolation and characterization of the multiple forms of human skeletal muscle triosephosphate isomerase.

1. Human skeletal muscle triosephosphate isomeras (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) was isolated and resolved by DEAE-cellulose chromatography into three major forms, A, B, and C, which comprise 97% of the total activity. The relative distribution was 25, 46 and 29% respectively. 2. The A and C forms are homodimers, alpha alpha and beta beta, and form B is the heterodimer, alpha beta. Reassociation studies from guanidinium chloride have indicated that A, B, and C are not conformers. Although these studies revealed the existence of two different chains, the amino acid analysis showed no significant variance. Since no differences were obsrved in Ouchterlony and Mancini tests or in immunotitration, the three fors are assumed to be immunologically identical. 3. The three forms have the same specific activity, Michaelis constants, pH optimum, activation energy, inhibition by metabolites and heat stability. Only with increasing ionic strength did the V and Km values differ. 4. The two poypeptide chains (alpha and beta) appear to be identical (amino acid composition, molecular weight and antigenity), and since the electrophoretic banding pattern changed with cell aging, it is concluded that the multiple forms of trisephosphate isomerase are the consequence of minor post-synthetic alteration(s) of form A.

Amino Acids↗
PubMed1980

A single amino acid substitution (Asp leads to Asn) in a phosphoglycerate kinase variant (PGK München) associated with enzyme deficiency.

The structural abnormality of the phosphoglycerate kinase variant, PGK München, associated with red cell enzyme deficiency and heat instability, was elucidated by a microscale peptide-mapping method. A single amino acid substitution, from aspartic acid in the normal enzyme to asparagine in the variant enzyme, was found. From the known amino acid sequence of normal human phosphoglycerate kinase, the substitution is in the aspartic acid residue located at 268th position from the NH2-terminal of the protein.

Amino Acid Sequence↗
PubMed1980

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."

Anemia, Hemolytic↗
PubMed1978

Isolation of phosphoglycerate kinases by affinity chromatography.

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.

Adenosine Diphosphate↗