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Phosphofructokinase (PFK; ATP: D-fructose-6-phosphate transferase, EC 2.7.1.11) was studied in human thyroid carcinomas (n = 16), follicular adenomas (n = 31) and normal thyroid tissue (n = 19). The specific activity in carcinomas (0.129 +/- 0.070) is significantly increased (p less than 0.001) in comparison with phosphofructokinase in normal thyroid tissue (0.028 +/- 0.009). No difference in phosphofructokinase activity seems to exist between follicular, papillary and undifferentiated carcinomas. Specific activities of follicular adenomas are rather heterogeneous. When these tumors were divided into three groups of increasing proliferative activity as judged by histopathological criteria, highest specific activities of phosphofructokinase were found in the group with the highest proliferative activity. The latter group resembles the enzyme activities found in carcinomas. On the other hand specific enzyme activities of the least active tissues were comparable to normal and different from carcinomas. Adenomas show the same isozyme composition as found in normal thyroid tissue. All three isozymes of PFK, M-(muscle)type, L-(liver)type and P-(platelet)type, were present. Phosphofructokinase from papillary carcinomas show a lesser degree of precipitation with anti-M antibodies. A higher amount of platelet type isoenzyme is found in papillary carcinomas compared to follicular and undifferentiated carcinomas. The influence of citrate, an inhibitor of phosphofructokinase, is in agreement with the isozyme composition.

Acceleration of glycolysis is, in general, a characteristic of neoplasia. Previous studies have shown that this increase in glycolysis is achieved by quantitative increases in the activities of the key regulatory enzymes, hexokinase, phosphofructokinase (PFK) and/or pyruvate kinase, which are often accompanied by isozymic alterations that facilitate glycolysis. In this study, we investigated the alterations in the activity, isozymic profile, and kinetic-regulatory properties of PFK from the medullary thyroid carcinomas of the rat, which represent a model for the neuroectodermally derived tumors in humans. Contrary to the expected, we found that undifferentiated tumors showed a decrease in the enzyme activity as compared to the highly differentiated tumors. This decrease in PFK activity was accompanied by an increase in the expression of the liver-type isozyme of PFK. The enzymes from the 2 tumor types showed no significant differences in their affinity and cooperativity toward the substrates, fructose 6-phosphate and adenosine triphosphate (ATP). However, the tumor PFKs showed major differences with respect to their behavior toward the allosteric regulators of the enzymes, ATP, citrate, and fructose 2,6-diphosphate; the latter is a recently discovered activator of the enzyme. The enzyme from the undifferentiated tumor was less sensitive to citrate inhibition, which was more readily reversed by cyclic adenosine 3':5'-monophosphate. In addition, it was less sensitive to ATP inhibition at low fructose 6-phosphate concentrations. More importantly, the enzyme from the undifferentiated tumors was more sensitive to the activation by fructose 2,6-diphosphate especially when inhibited by citrate and ATP. The altered regulatory properties of the enzyme from the undifferentiated tumors most probably reflect its altered isozymic composition, i.e., increase in the liver-type isozyme. The preferential expression of the liver-type isozyme by undifferentiated and rapidly replicating cancer cells may be explained in terms of the unique regulatory properties of this isozyme. Although the concentrations of fructose 2,6-diphosphate were comparable in these 2 tumor types, the higher sensitivity of the liver-type PFK to activation by this compound may permit accelerated glycolytic flux observed in undifferentiated tumors, despite a decrease in total PFK activity.

The enzymes hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were studied in rat lung during development starting at day 16 of gestation (day-6) until 5 days after birth. During gestation, the activities of hexokinase type II, enolase and pyruvate kinase decreased and reached adult values at birth or shortly thereafter. Hexokinase type I remained relatively constant and the decrease of soluble type II hexokinase was compensated for by an increment of particle-bound hexokinase starting at day 20 of gestation until birth. In contrast, phosphofructokinase activity increased until day 20 of gestation followed by a rapid fall in activity until 2 days after birth. Except for hexokinase no isoenzyme shifts were observed in the period of observation. The results are discussed with respect to the proposed relationship between glycogen breakdown and surfactant synthesis during the perinatal period and suggest a regulatory role for phosphofructokinase in this process.

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.

Pyruvate kinase isozymes were studied in normal brain tissue (both fetal and adult) and in meningiomas and malignant gliomas. In fetal brain five different forms could be detected by electrophoresis (K4, K3M, K2M2, KM3, and M4). In adult brain the M4-type, K3M hybrid, and K4-type are present; the M isozyme is largely predominant. Alanine inhibition of pyruvate kinase is in agreement with the electrophoretic pattern. Pyruvate kinase from fetal brain and brain of a newborn is more inhibited compared with pyruvate kinase from adult brain. The Lineweaver-Burk plots for pyruvate kinase from fetal brain and brain of the newborn are nonlinear due to the presence of hybrids. Pyruvate kinase from meningiomas and malignant gliomas is strongly inhibited by alanine. Electrophoresis proved the presence of mainly K4 type and the hybrid K3M, which is in agreement with the alanine inhibition. Determination of the Km's for phosphoenolpyruvate supports this conclusion. The determination of the alanine inhibition of pyruvate kinase may be a diagnostic tool in surgery for gliomas.

Eleven lysosomal enzyme activities were tested in lymphocytes from normal individuals and patients with chronic lymphocytic leukemia. The activities of all enzymes, except acid phosphatase, were significantly lower in the leukemic lymphocytes (p less than 0.001). In addition the activities were tested in purified T- and non-T lymphocytes and in monocytes. For most enzymes the activities are similar in the three cell types, except for alpha-D-galactosidase, arylsulphatase B and alpha-D-glucosidase, which are lower in T lymphocytes, and N-acetyl-beta-D-glucosaminidase which is lower in non-T cells. In T and B lymphoblastic cell lines the activities are within the range for leukemic lymphocytes. No differences were found between the T and the B cell lines.

A new procedure for the assay of protein tyrosine kinase, based on the detection of phosphorylated tyrosyl residues by using monoclonal antibodies to phosphotyrosine, is described. After incubation of a protein tyrosine kinase sample with the substrates poly-(GluNa,Tyr)4:1 and unlabeled ATP an aliquot of the reaction mixture is transferred to a polyvinylidene difluoride membrane. The extent of tyrosine phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection by the immunogold silver staining procedure. The signal is quantified by densitometry. The assay is linear with time and is quantitative in a wide range of sample protein concentrations. Its sensitivity allows the kinetic characterization of protein tyrosine kinases at low substrate concentrations, whereas on the other hand the avoidance of radioactivity enables the use of high ATP concentrations as well. Protein tyrosine kinase activities of human breast carcinomas and normal breast tissues measured with this method correlated well with the conventional assay, in which the incorporation of [32P]phosphate is measured by TCA precipitation and liquid scintillation counting. Compared to the latter, the new assay is at least as sensitive and accurate and harbors the advantage of the avoidance of radioactivity, thus enabling one to perform a large number of protein tyrosine kinase assays simultaneously.

Deficiency of purine nucleoside phosphorylase (PNP) was detected in a 3-yr-old boy who was admitted for investigation of a behavior disorder and spastic diplegia. The urinary excretion of purines, analyzed by high-performance liquid chromatography, showed the presence of large amounts of (deoxy)inosine and (deoxy)guanosine and low uric acid levels. Analysis of the (deoxy)nucleotide pools of erythrocytes showed elevated levels of deoxyguanine nucleotides and NAD and decreased guanine nucleotides. PNP activity in red blood cells was 0.1-0.5% of normal on two occasions and undetectable on four later measurements. Furthermore no immunoreactive material could be detected in his red cell lysate using an anti-PNP antiserum. PNP activities in the red cells of the patient's parents were 35 and 50% of normal. The presence of (minor) residual PNP activity in the patient enabled the investigation of some enzyme properties after partial purification. No abnormalities could be detected in substrate affinity for inosine, heat stability, and electrophoretic properties. In the heterozygous parents no signs of a mutant enzyme could be found. The molecular specific activities of the parental enzymes were also normal, indicating that no immunoreactive material attributable to inactive-mutant enzyme subunits was present. A striking feature of the patient is the prevailing neurologic abnormalities presumably caused by the metabolic disorder. A severe lymphopenia exists; however, clinical symptoms of an immune deficiency did not become apparent until the age of 4 yr.

The pyruvate kinase isozymes M1 and M2 are structurally and immunologically closely related. To obtain an antibody which discriminates between these two forms, a synthetic tetradecapeptide with a sequence specific for pyruvate kinase type M2 from rats was constructed. Antisera from rabbits, immunized with this peptide, reacted specifically with the M2-type holoenzyme of both rat and human origin, and did not cross-react with the M1-type isozyme. This was established by immunoblot analysis, both under dissociating and non-dissociating conditions.

In this study, we investigated the alterations in the activity, subunit profile, and kinetic regulatory properties of phosphofructokinase (PFK) from human gliomas compared with those from normal human brain. Gliomas showed a decrease in the enzyme activity as compared to normal brain. This decrease in PFK activity was accompanied by a relative increase in the expression of the liver type subunit of PFK. The enzymes from the tumor and normal brain showed no significant differences in their affinity toward the substrate fructose 6-phosphate. However, tumor and normal brain PFK showed major differences with respect to their behavior towards citrate and fructose 2,6-bisphosphate. The enzyme from the gliomas was less sensitive to citrate inhibition. More importantly, the enzyme from the tumor was more sensitive to the activation by fructose 2,6-bisphosphate. In addition, we found that in gliomas the L-type subunit could be phosphorylated, most probably by a cyclic AMP-independent protein kinase. This phosphorylation could not be detected in normal human brain. It is proposed that the preferential expression of the liver type subunit by undifferentiated cancer cells may be explained in terms of the unique regulatory properties of this isozyme.

gamma-Enolase [one of the three possible subunits of the dimeric enzyme enolase (EC 4.2.1.11)] has been reported as a marker for human neurons, neuroendocrine cells and tumors derived from these cells. In recent years, however, its presence has been reported in nonneuronal tumors. For employment in the histopathologic diagnosis of tumors of the nervous system, exact knowledge of the enolase isoenzyme patterns occurring in these tumors is a prerequisite. In human gliomas, the presence of varying quantities of gamma-enolase has been demonstrated. The present study examines the enolase isoenzyme pattern in human cerebral metastases of various primary progeny, using electrophoresis of tumor tissue extracts as well as immunohistochemistry. Additionally, a number of primary tumors of nonneuroepithelial tissue was examined by immunohistochemistry. The presence of gamma-enolase was demonstrated in a significant number of brain metastases. A relation between enolase isoenzyme pattern and survival after operation for brain metastasis could not be found. For histopathologic diagnosis of tumors of the adult human central nervous system, analysis of the enolase isoenzyme pattern is not reliable.

Erythrocytes were separated by age using a combination of density centrifugation and counterflow centrifugation and tested for basal activity of the hexose monophosphate shunt (HMP-shunt) as well as the methylene blue-stimulated maximal capacity by measuring CO2 production. No significant differences were found in basal HMP-shunt activity, but the maximal methylene blue-stimulated activity of old erythrocytes reached only half of the activity of the total cell population. The maximal HMP-shunt activity showed a significant correlation with hexokinase activity, but not with glucose-6-phosphate dehydrogenase activity in all but the youngest cells. The sensitivity to oxidative stress was tested by measuring the kinetics of pyruvate kinase isolated from erythrocytes incubated in presence and absence of methylene blue. Pyruvate kinase kinetics were affected more in the old cell population than in the total cell population: the K0.5 for phosphoenol-pyruvate increased four times in the unseparated cells and eight times in old cells.

Gamma-enolase (one of the three possible subunits of the dimeric enzyme enolase (EC 4.2.1.11)) has been reported as a marker for human neurons. Studies investigating the presence of gamma-enolase in human gliomas have given conflicting results, but a definite finding is important for further studies of the biology of these tumors and the possible use of gamma-enolase as a marker for tumors originating in nervous tissue or for neuronal damage. Using electrophoresis of tumor tissue extracts as well as immunohistochemistry the authors have demonstrated the presence of gamma-enolase in human gliomas. Analysis of the gamma-enolase content in the plasma of patients with brain neoplasms further revealed that, although this enzyme may be present in the tumor itself, its concentration in blood is not a reliable marker for a tumor of the human central nervous system.

Human erythrocyte hexokinase (EC 2.7.1.1) is inhibited competitively with respect to Mg-ATP2- by uncomplexed Mg2+ (Ki = 16--18 mM) and ATP4- (Ki = 1.6 mM). No real activation by low concentrations of Mg2+ could be detected and no allosteric behaviour was observed under the conditions tested. The temperature dependence of the enzyme was studied in relationship to the presence of Mg2+ or ATP4-. At equal concentrations of Mg2+ and ATP4- a break in the Arrhenius plot was observed at 27.5 degrees C, the higher temperature form of the enzyme having the lower activation energy. This break point in the Arrhenius plot was shifted to 36 degrees C in the presence of 5 mM Mg2+. A straight-line relationship was observed in the presence of 2.5 mM ATP4-. The Km for Mg-ATP2- showed a linear increase at temperatures over about 36 degrees C independent of the presence of Mg2+ or ATP4-. The nature of these phenomena is discussed.

Riboflavin status was studied in 156 older healthy people living at home in The Netherlands by assaying erythrocyte glutathione reductase (with and without FAD). The average activation coefficient of glutathione reductase was found to be 1.42 +/- 0.19. 7% of the population studied showed cataract (based on the eye examination). It seems that no correlation exists between cataract and riboflavin deficiency.

In this study pyruvate kinase, hexokinase and aldolase are investigated in two types of embryonal tumors, neuroblastomas and medulloblastomas; the results are compared with similar studies in gliomas. The activities of hexokinase and pyruvate kinase are significantly decreased in neuroblastomas. In neuroblastoma and medulloblastoma all five forms of pyruvate kinase (K4, K3M, K2M2, KM3 and M4) are present. In contrast, the gliomas investigated are characterized by the presence of mainly K4 and a little K3M. In neuroblastomas, medulloblastomas and gliomas, hexokinase type I is present; in addition, hexokinase type II is present in two medulloblastomas. Aldolase A is the predominant isozyme in all tumors investigated; this is in contrast with normal nervous tissue. It can be concluded that the isozyme characteristics especially of pyruvate kinase from neuroblastomas and medulloblastomas are comparable with similar findings in retinoblastoma; these findings support the hypothesis that these three tumors have a common embryonic origin.

The activities of hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase were studied in breast cancer tissues, in comparison to benign breast disease and normal breast tissues. The enzyme activities in breast cancer were significantly increased compared to normal and benign breast tissues (p less than 0.001). Also the increase in activity in benign disease compared to normal was statistically significant (p less than 0.001). Within the group of benign diseases, fibroadenomas could be distinguished from fibrocystic disease, the former generally showing higher activities compared to the latter (p less than or equal to 0.05). Carcinoma subgroups, classified according to their histology, could not be recognized enzymologically. In addition, isozyme composition of pyruvate kinase and enolase was studied. We did not find a significant shift towards K type pyruvate kinase expression in benign disease compared to normal breast tissues. Also fibroadenomas did not differ from fibrocystic disease. However, the amount of K type pyruvate kinase in carcinomas proved to be significantly higher in comparison to benign disease and normal breast tissues (p less than 0.001). Expression of alpha gamma-enolase in normal breast tissue was virtually absent. In benign disease only a minority of specimens did show the hybrid alpha gamma-enolase. Nearly all carcinomas had alpha gamma-enolase expression and in 20% of the carcinomas gamma gamma-enolase could be detected (so-called neuron-specific enolase). By discriminant analysis, the function giving the best discrimination compared to the histological data was based on natural logarithm aldolase and the total of gamma-enolase subunits. Contrary to expectation, the regulator enzymes of glycolysis; i.e., hexokinase, phosphofructokinase and pyruvate kinase were not included in this discriminant function. The best fit produced a 90% correct classification in both benign and malignant disease. If these findings are confirmed to a larger series, the discrimination is sufficiently strong to form the basis of a clinically useful tool.

We have isolated variants of L1210 cells (L1210B) expressing, in addition to the "classical" high affinity/low capacity system for reduced folate uptake, high levels of a membrane-associated folate binding protein. This folate binding protein was expressed in L1210 cells grown at low physiological folate levels (less than 0.5 nM), but down-regulated after transfer in standard high folate (2 microM) medium. The binding capacity of L1210B cells for [3H]folic acid and [3H]-methotrexate was identical (5-11 pmol/10(6) cells) but affinities were different. The affinities relative to folic acid were 0.5 for 5-methyltetrahydrofolate, 0.25 for 5-formyltetrahydrofolate, 0.08 for 10-ethyl-10-deazaaminopterin, and 0.05 for methotrexate, respectively. L1210B cells exposed to low extracellular concentrations of [3H]folic acid (25 nM) accumulated 15 pmol [3H]folic acid/10(7) cells over a 5-h period. [3H]Folic acid accumulation by wild-type L1210 cells could not be demonstrated under these conditions. The folate binding protein in L1210B cells could be specifically and covalently labeled at 4 degrees C with a N-hydroxysuccinimide ester of [3H]-methotrexate or [3H]folic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent-solubilized membrane proteins showed a major labeled band with Mr 42,000-44,000.

A glucose-6-phosphate isomerase deficiency is described in an Arab boy suffering from chronic hemolytic anemia. The patient was probably true homozygous for the defect. The residual enzyme activity in his red blood cells (RBC) was approximately 30% of normal. The most striking enzyme abnormality observed was an extreme heat lability: upon incubation at 45 C, greater than 90% of activity was lost within 15 min. Furthermore, an increased affinity for the substrate glucose-6-phosphate was shown. The lability of the enzyme was also shown to exist in vivo by separating the patient's RBC into four fractions of different cell age by centrifugation on a discontinuous density gradient. This in vivo lability of the enzyme is believed to be the main cause of the hemolytic diathesis. Remarkably, the residual activity of the enzyme in the RBC of obligate heterozygotes was comparable to that in the patient. However, their enzyme activity was only slightly more labile than that in normal RBC and consequently no signs of hemolysis were noticed.

The membrane impermeant protein cross-linker 3,3'-dithiobissulfosuccinimidyl propionate (DTSSP) is a well-known inhibitor of human erythrocyte band 3-mediated inorganic anion transport. We observed that DTSSP is also a potent inhibitor of reduced folate/methotrexate transport in human CCRF-CEM leukemia cells. An interaction of DTSSP with the reduced folate/MTX is substantiated by findings that: (a) like MTX transport itself, the concentration of DTSSP required for half-maximal inhibition of [3H]methotrexate transport varied substantially with the anionic composition of the external medium. In a saline buffer and an anion-deficient buffer the I50 values were 7 and 1 microM, respectively; (b) saturation of the carrier with 1-5 microM methotrexate completely protected the transport system from interaction by DTSSP; (c) methotrexate transport activity in DTSSP-treated cells could be restored after cleavage of the disulfide bond in DTSSP under mild reducing conditions; and (d) pretreatment of cells with DTSSP reduced the incorporation of [3H]methotrexate after labeling with an N-hydroxysuccinimide ester of [3H]methotrexate (NHS-MTX), another potent inhibitor of methotrexate transport. Comparison of DTSSP- and NHS-MTX-induced inhibition of methotrexate transport showed that DTSSP inhibition, in contrast to NHS-MTX inhibition, was (a) less potent, (b) dependent on buffer conditions, (c) reversible by reducing agents, and (d) required only a very low molar ratio of methotrexate over DTSSP to afford maximal protection.

Pyruvate kinase from anaplastic medullary thyroid carcinomas contains predominantly K-type subunits, whereas pyruvate kinase from differentiated medullary thyroid carcinomas consist of M- and K-type subunits in about equal proportion. In order to analyse the incorporation of phosphate in the respective isozymes after endogenous phosphorylation of cytosolic extracts with [32P]ATP, homotetrameric isozymes as well as heterotetrameric hybrids of differentiated tumors were resolved by affinity chromatography on Blue-Sepharose CL-6B and, if necessary, further purified by immunoprecipitation. SDS-polyacrylamide gel electrophoresis of purified isozymes and subsequent autoradiography showed the incorporation of phosphate in the K4-type isozymes, but not in the other isozymes. The phosphorylation appeared to be cAMP-independent and occurred on a serine residue.

GLycolytic enzymes were studied from normal human retinas (both fetal and adult) and from retinoblastomas of eight patients and an established retinoblastoma cell line. No significant differences were found between the enzyme activities in the tissues investigated except for hexokinase and pyruvate kinase, which were significantly decreased in the tumor cells. In fetal retina, five different forms of pyruvate kinase could be detected by electrophoresis (K4, K3M, K2M2, KM3, and M4). In adult retina the K4 isozyme is almost absent, while in retinoblastoma the M4 isozyme is hardly present. In the retinoblastoma cell line, the M4 isozyme is completely absent. Alanine inhibition of pyruvate kinase from the retinoblastoma cell line is more inhibited compared to the pyruvate kinase of fetal retina and retinoblastoma and is even more inhibited compared to adult retina. Electrophoresis of aldolase from adult retina revealed the presence of all potential A-C hybrids (A4, A3C, A2C2, AC3, and C4). Fetal retina, however, is characterized by the predominance of the A type. The same patterns were observed in the retinoblastoma cell line and retinoblastoma. However, in other brain tumors, e.g., gliomas of adults, a five-membered A-C hybrid set is found. Electrophoresis of hexokinase from normal fetal and adult retina revealed the predominance of hexokinase type I; retinoblastoma and retinoblastoma cell line are both characterized by the presence of considerable amounts of hexokinase type II. The isozyme shifts in retinoblastoma result in an enzyme pattern identical to that of fetal retina except for the presence of hexokinase type II.

K-type pyruvate kinase was purified from human kidney by immunoadsorbant chromatography. Monoclonal antibodies secreting hybridomas were made using conventional techniques. Two clones were established which produced antibodies against K-type not cross-reacting with the other pyruvate kinase isoenzymes, named the M, L and R-types. The specificity of the monoclonal antibodies was proven by enzyme-linked immunosorbent assay, immunoprecipitation and immunoblotting experiments. The M- and K-isoenzymes are produced from the same gene probably by alternative splicing, and all differences between both enzymes originate from one exon coding for 45 amino acids (Noguchi et al. J. Biol. Chem. 261, 13807-13812 (1986]. The monoclonal antibodies are specific for K-type under denaturing conditions. Thus, it is likely that these antibodies recognize (a) continuous epitope(s), of which at least some amino acids are coded in the K-specific exon. The monoclonal antibodies could be successfully used in immunohistochemical studies. Neurons and astrocytes in brain, Kupffer cells in liver, connective tissue cells and vascular smooth muscle cells showed immunoreactivity. However, striated muscle cells in skeletal muscle and heart and hepatocytes were not immunoreactive. Other types of glial cells, e.g., oligodendrocytes and microglia, so far studied, showed no reaction either.

In primary human brain tumours a shift occurs in the synthesis of isoenzymes of pyruvate kinase from the M towards the K-type. In astrocytomas, oligodendrogliomas and glioblastomas, which were localised in the cerebral hemispheres of adult patients over 20 years of age, the shift correlated well with histological grading and growth rate as observed in postoperative survival. Gliomas of adults, localised in midline structures, as well as childrens gliomas were characterised too by a strong shift from M towards the K type. However, in these tumours, less correlation with histological grading and growth rate was found. The isoenzyme shift can be rapidly demonstrated with an alanine inhibition test. The application of this assay may have a diagnostic value during operation for gliomas in grading of malignancy in adults as well as demarcation of the resection of gliomas in all age groups. The test can be performed within 10-15 min and can thus fit easily into a surgical procedure. A case report is presented for illustration.

In 6 patients with breast cancer - of whom specimens of the primary tumor as well as one of its metastases were available for examination - we demonstrated intratumoral and intertumoral heterogeneity in expression of activity of the glycolytic enzymes hexokinase, phosphofructokinase, aldolase, enolase and pyruvate kinase. Heterogeneity also existed in isozyme composition of pyruvate kinase. The transition of the tumors towards normal surrounding breast tissue showed either a sharp drop in activity, or a gradual decrease in activity, corresponding to pushing margins or infiltrative growth of the tumor as was demonstrated by histologic examination of these specimens. Likewise, the shift towards expression of K isozyme of pyruvate kinase in breast cancer compared to normal breast tissue could be demonstrated.

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.