Gerard E.J. Staal

Purine Nucleoside Phosphorylase Deficiency Associated with Selective Cellular Immunodeficiency

We studied a 15-month-old girl who had normal T-cell and B-cell immunity at birth, after which a gradual decrease in T-cell immunity developed. This selective cellular immunodeficiency was inherited as an autosomal recessive trait: two older sisters had the same immunodeficiency. Adenosine deaminase activity was present in erythrocytes and lymphocytes of the patient, parents and a healthy brother. Purine nucleoside phosphorylase activity was not found in the patients erythrocytes and lymphocytes (the parents and brother had intermediate values, indicating that the enzyme deficiency too was inherited as an autosomal recessive trait). Analysis of serum and urine from the patient and of serum from her two deceased sisters showed high levels of inosine and guanosine in addition to hypouricemia and hypouricosuria. The bone marrow was megaloblastic, and the blood hypochromic microcytic. The patient had spastic tetraparesis. Intoxication of the T lymphocytes after birth by metabolic products may explain the progressive cellular immunodeficiency.

Glycolytic Enzyme Activities in Breast Cancer Metastases

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase were studied in breast cancer metastases occurring at various sites and compared with the enzyme activities in a series of primary breast cancers. The activities of all enzymes studied were significantly higher in the metastases compared to the primary tumors (p less than or equal to 0.05). However, no changes in the isoenzyme patterns of enolase and pyruvate kinase were observed when the metastases were compared with primary breast cancers. Differences in location of the metastases did not lead to differences in enzyme activities. Our data suggest an association of an increasing rate of glycolysis with tumor progression.

Characterization of α-L-fucosidase from two different families with fucosidosis

Abstract 1. 1. Two different families with a different type of fucosidase deficiency are described. 2. 2. In the first family the activity of α-L-fucosidase in leucocytes of two patients with fucosidosis type I was about 4 to 8% of the normal value. The activity of α-L-fucosidase in the leucocytes of the father and the mother are in the heterozygote range, while a sister of the propositus showed normal values. 3. 3. The activity of α-L-fucosidase of the propositus from urine, serum and liver were also severely decreased. The activity of α-L-fucosidase in the urine of the parents and the healthy sister of the propositus were about 5% of the mean normal value. However in the serum these values were above 50%. 4. 4. The K M value for α-L-fucosidase from leucocytes of the patient was increased about 10 times and in serum this value was even higher. The K M values from the enzyme of the parents were in the normal range. 5. 5. The abnormal enzyme from the propositus is unique in its thermal behaviour since after heating its activity increased. 6. 6. In the second family the activity of α-L-fucosidase in the leucocytes of the patient is about 30% of the mean normal value, while the arylsulphatase A activity is also decreased (25% of the mean normal value). 7. 7. The activity of α-L-fucosidase from the leucocytes of the father and the healthy brother are about 50% of the mean normal level, while the enzyme of the mother showed a normal activity. 8. 8. The α-L-fucosidase activity in the urine and the liver of the propositus is also decreased. The serum enzyme activity however was in the normal range.

Glycolytic Enzymes in Breast Cancer, Benign Breast Disease and Normal Breast Tissue

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyru-vate kinase were studied in breast cancer tissues, in comparison to benign breast disease and normal breast tissues. The e

Regulation of human erythrocyte hexokinase: The influence of glycolytic intermediates and inorganic phosphate

Human erythrocyte hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was inhibited competitively with respect to MgATP2- by glucose-6-P (Ki - 10.8 muM) and fructose-6-P (Ki = 160 muM). Low concentrations of inorganic phosphate were competitive with respect to glucose-6-P and fructose-6-P, although higher concentrations of Pi were not able to overcome completely the inhibition by the hexose phosphates. The results are consistent with a model in which hexokinase exists in equilibrium either as free or phosphate-associated enzyme, the latter having a reduced but still substantial affinity for hexose phosphate. An alternative explanation could be found in the presence of two different enzymes, one with a high affinity for glucose-6-P being sensitive to regulation by Pi, one with a lower affinity for glucose-6-P being insensitive to Pi. A similar but less pronounced effect of Pi, was found on the inhibition by 2,3-diphosphoglycerate (Ki = 4.0 mM). Pi in the absence of inhibitor was also a competitive inhibitor with respect to MgATP2- (Ki = 20 mM). Furthermore a competitive inhibition with respect to MgATP2- was found by fructose 1,6-diphosphate (Ki = 4.3 mM), glycerate-3-P (Ki = 3.8 mM), glycerate-2-P (Ki = 12.5 mM), MgADP- (Ki = 1.0 mM) and MgAMP (Ki = 1.7 mM).

Alkyllysophospholipid ET-18-OCH3 acts as an activator of protein kinase C in HL-60 cells

HL‐60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET‐18‐OCH3 on the activity of protein kinase C. HL‐60 cells were incubated with ET‐18‐OCH3 at a concentration of 20 μg/ml for 4 h. After the incubation the membrane fraction of the HL‐60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser25]‐protein kinase C (19–31) as a protein kinase C specific substrate. The activity of the membrane‐bound protein kinase C was increased in HL‐60 cells treated with ET‐18‐OCH3 compared to untreated HL‐60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12‐myristate 13‐acetate. In contrast, solubilized protein kinase C from HL‐60 cells could be inhibited or stimulated in vitro by ET‐18‐OCH3, dependent on the mode of addition of ET‐18‐OCH3 and phospholipids.

Functional interaction between the epidermal growth factor receptor and c-Src kinase activity

To study the relationship between the tyrosine kinase c‐Src and the epidermal growth factor receptor (EGF‐R), we used the breast cancer cell line ZR75‐1, which was transfected with the EGF‐R. The EGF‐R transfected cell line expressed 60 times more EGF‐R than a control cell line transfected with the empty vector. In the presence of EGF, the EGF‐R over‐expressing cell line grew much faster than the control cell line. Both cell lines expressed approximately equal amounts of c‐Src. However, the cell line over‐expressing the EGF‐R showed a twofold enhancement of c‐Src kinase activity after EGF stimulation. The activation of c‐Src kinase by EGF was confirmed in other EGF‐R expressing cell types.

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome.

SummaryA patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent Km for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the Km of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patients mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patients family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.

L-α-Alanine Inhibition of Pyruvate Kinase from Tumors of the Human Central Nervous System

There is ever-increasing literature pointing out that gene expression in tumour cells is different from that in normal cells from which or in which the tumour originates. In general, neoplasia is characterized by misprogramming of protein synthesis (16).

Purification and some properties of human erythrocyte hexokinase.

1. Human erythrocyte hexokinase (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified 50 000--100 000-fold with a final specific activity of about 25--50 units/mg protein using gel-filtration, ion-exchange chromatography and affinity chromagraphy. 2. After isoelectrofocusing ofthe preparation one major protein band could be detected besides a minor band. THe isoelectric point of the major protein band was found to be 4.7. 3. After purification the enzyme could be stabilized in a medium containing inorganic phosphate, glucose, glycerol and mercaptoethanol. 4. The molecular weight was determined by gel-filtration and was found to be 132 000+/-8000. 5. The enzyme shows a broad pH optimum ranging from 7.0 to 8.4. 6. The kinetic behavior of the purified enzyme at 37 degrees C was somewhat different from the normal Michaelis-Menten kinetics due to its instability. The affinity constants were 0.048--0.080 mM for glucose and 0.57--1.0 mM for Mg-ATP. 7. The enzyme was specific for Mg- ATP as the nucleotide substrate. Mg-UTP, Mg-ITP,Mg-GTP and Mg-CTP were not converted to corresponding diphosphates. Several hexoses could be phosphorylated by the enzyme. Mannose could be phosphorylated at the same rate as glucose, although the affinity for the enzyme was lower (5m=0.60mM). Much lower rates and lower affinities were found with 2-deoxy-D-glucose (5m=1.0mM), D(+)-glucosamine (5m=4.5 mM) and fructose (5m=10 mM). N-acetyl-D-glucosamine , galactose andsorbose were not phosphorylated at all.