A study of PKM2, PFK-1, and ANT1 expressions in cervical biopsy tissues in China.

This present study explored the association of Pyruvate Kinase isozyme M2 (PKM2), Phosphofructokinase 1 (PFK-1) and Adenine nucleotide translocator 1 (ANT1) with cervical carcinoma. A case-control method was designed by the collected 95 cervical biopsy samples, which were divided into 30 controls and 60 cases. Cases were subdivided into mild cervical carcinoma (MCC-25), intermediate cervical carcinoma (ICC-20), and severe cervical carcinoma (SCC-20) by method of cervical pathology. The expression of PKM2, PFK-1, and ANT1 was examined by methods of immunohistochemistry and western blotting (WB). The results showed that the positive proportions of PKM2 and PFK-1 in case group were higher than that of control, and the increased positive proportions of PKM2 and PFK-1 were also revealed with the order of Control, MCC, ICC, SCC (P<0.05). Further, the results of WB confirmed the enhanced expressions of PKM2 and PFK-1 in case group and the increasing trend of PKM2 and PFK-1 expressions in Control, MCC, ICC, and SCC groups. In addition, the WB result of ANT1 showed a lower level of expression in SCC group, while the positive proportion of ANT1 was not significant between cases group and control. In conclusion, PKM2 and PFK-1 genes are associated closely with cervical carcinoma. The enhanced expressions of PKM2 and PFK-1 indicate one developing signal of cervical carcinoma.

Regulatory properties of 6-phosphofructokinase and control of glycolysis in boar spermatozoa.

Glycolysis is crucial for sperm functions (motility and fertilization), but how this pathway is regulated in spermatozoa is not clear. This prompted to study the location and the regulatory properties of 6-phosphofructokinase (PFK, EC 2.7.1.11), the most important element for control of glycolytic flux. Unlike some other glycolytic enzymes, PFK showed no tight binding to sperm structures. It could readily be extracted from ejaculated boar spermatozoa by sonication and was then chromatographically purified. At physiological pH, the enzyme was allosterically inhibited by near-physiological concentrations of its co-substrate ATP, which induced co-operativity, i.e. reduced the affinity for the substrate fructose 6-phosphate. Inhibition by ATP was reinforced by citrate and H+. Above pH 8, PFK lost all its regulatory properties and showed maximum activity. However, in the physiological pH range, PFK activity was very sensitive to small changes in effectors. At near-physiological substrate concentrations, PFK activity requires activators (de-inhibitors) of which the combination of AMP and fructose 2,6-bisphosphate (F2,6P2) was most efficient as a result of synergistic effects. The kinetics of PFK suggest AMP, F2,6P2, H+, and citrate as allosteric effectors controlling PFK activity in boar spermatozoa. Using immunogold labeling, PFK was localized in the mid-piece and principal piece of the flagellum as well as in the acrosomal area at the top of the head and in the cytoplasmic droplets released from the mid-piece after ejaculation.

Activity, distribution and regulation of phosphofructokinase in salivary gland of rats with streptozotocin-induced diabetes

Apesar de existirem muitos estudos sobre a influência do diabetes nas glândulas salivares, esses apresentam resultados conflitantes. Neste estudo, a regulação da enzima fosfofrutoquinase-1 (PFK-1) foi estudada utilizando-se glândulas salivares de ratos. O diabetes foi induzido por uma única injeção intraperitonial de estreptozotocina (60 mg/kg peso corporal) em ratos (180-200 g). Os animais foram sacrificados 30 dias após a indução do diabetes e utilizaram-se as glândulas submandibular e parótida. A hiperglicemia foi avaliada por determinação da glicemia sanguínea. A distribuição da PFK-1 entre frações solúvel e ligada, concentração de fosfato na PFK-1, concentração de frutose-2,6-bisfosfato e a atividade da enzima PFK-2 foram determinadas. O cálculo do peso glandular relativo mostrou um aumento na glândula parótida de ratos diabéticos comparados ao controle, o que não ocorreu na glândula submandibular. A atividade da PFK-1 expressa por glândula não mostrou variação entre animais diabético e controle. Contudo, considerando a atividade específica, a fração solúvel da enzima mostrou aumento de 50% com relação ao controle e a fração ligada ao citoesqueleto um aumento de 84% com relação ao controle. Na glândula parótida não foi observada diferença na atividade específica entre os grupos diabético e controle. Por outro lado, a atividade por glândula da fração solúvel aumentou nos animais diabéticos. A concentração de fosfato da PFK-1 aumentou nas glândulas submandibular e parótida nos animais diabéticos. Tanto a concentração de frutose-2,6-bisfosfato quanto a forma ativa da PFK-2 mostraram redução nas glândulas salivares. Concluindo, o aumento na atividade da PFK-1 observado nas glândulas salivares de ratos com diabetes induzida por estreptozotocina não parece ser modulado pela frutose-2,6-bisfosfato.

Different forms of obesity as a function of diet composition.

OBJECTIVE: To characterize the phenotype of obesity on a high-carbohydrate diet (HCD) as compared to a high-fat diet (HFD) or moderate-fat diet (MFD). METHODS AND PROCEDURES: In four experiments, adult Sprague-Dawley rats (275-300 g) were maintained for several weeks on a: (1) HFD with 50% fat; (2) balanced MFD with 25% fat; or (3) HCD with 10% fat/65% carbohydrate. Then, based on the amount of body fat accumulated in four dissected fat pads, the animals were subgrouped as lean (lowest tertile) or obese (highest tertile) and characterized with multiple measures. RESULTS: The obese rats of these diet groups, with 70-80% greater body fat than the lean animals, exhibited elevated levels of leptin and insulin and increased activity of lipoprotein lipase in adipose tissue (aLPL), with no change in muscle LPL. Characteristics common to the obese rats on the HFD or MFD, but not seen on the HCD, were hyperphagia, elevated circulating levels of triglycerides (TG), nonesterified fatty acids (NEFA) and glucose, and a significant increase in beta-hydroxyacyl-CoA dehydrogenase (HADH) activity in muscle, reflecting its greater capacity to metabolize fat. This was accompanied by a significant increase in expression of the peptide, galanin (GAL), in the paraventricular nucleus (PVN), as measured by in situ hybridization and real-time quantitative PCR, and also in GAL peptide immunoreactivity. These measures of GAL were consistently, positively correlated with circulating TG levels and also with HADH activity in muscle. In contrast to these fat-associated changes, rats that became obese on an HCD maintained normal caloric intake and levels of TG, NEFA, and glucose. They also showed no change in PVN GAL mRNA or peptide. Instead, they exhibited a significant reduction in HADH activity compared to the lean animals, along with increased activity of phosphofructokinase in muscle, a key enzyme in glycolysis. CONCLUSION: Specific characteristics of obesity, including expression of hypothalamic peptides, are dependent upon diet composition. Whereas obesity on an HFD is associated with hyperphagia and elevated lipids, fat metabolism in muscle, and fat-stimulated peptides such as GAL, obesity on an HCD with a similar increase in body fat shows none of these characteristics and instead exhibits a metabolic pattern in muscle that favors carbohydrate over fat oxidation. These results suggest the existence of multiple forms of obesity with different underlying mechanisms that are diet dependent.

A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme.

Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.

[Metabolic activity of the external intercostal muscle of patients with COPD]. / Actividad metabólica del músculo intercostal externo en pacientes con EPOC.

INTRODUCTION: The external intercostal muscle is a relevant contributor to ventilatory work in situations of overloading. Like other respiratory muscles, the external intercostal muscle seems to undergo a process of structural remodeling to adapt to a situation of functional disadvantage. However, findings from published studies of morphology have differed to a certain degree. On the one hand, the proportion of fibers involved in anaerobic metabolism increases; on the other hand, the number of capillaries also increases, an occurrence that would facilitate aerobic metabolism. OBJECTIVE: This study was designed to analyze the activity of several key enzymes involved in the principal metabolic pathways in the external intercostal muscles of patients with COPD. METHODOLOGY: We studied 6 patients with COPD (65 +/- 8 years, BMI 23 +/- 3 kg/m2, FEV1 51 +/- 9% ref, RV 184 +/- 38% ref, PaO2 81 +/- 10 mmHg) and 6 control subjects matched for age and anthropometric variables but with normal lung function. External intercostal muscle samples were taken from each patient (fifth intercostal space, non-dominant side). The samples were treated by conventional spectrophotometry to determine enzyme activity as follows: citrate synthase (CS, Krebs cycle), phosphofructokinase (PFK, by common glycolysis), lactate dehydrogenase (LDH, anaerobic glycolysis) and creatine phosphokinase (CPK, use of energy reserves). RESULTS: Patients with COPD showed greater PFK enzyme activity (93 +/- 25 versus 44 +/- 9 micromol/min/g of fresh weight; p = 0.001) and LDH (308 +/- 42 versus 231 +/- 29 micromol/min/g; p < 0.01) than did control subjects. However, CS and CPK activity was similar in both groups (82 +/- 31 versus 90 +/- 20 micromol/min/g and 4017 +/- 1734 versus 3048 +/- 464 micromol/min/g, respectively), although the latter displayed noteworthy dispersion of values among COPD patients, with levels in some patients being three-fold greater than in controls. RV was directly related to glycolytic enzyme activity (with PFK, r = 0.716, p < 0.01; with LDH r = 0.697, p < 0.05) and PFK and LDH also correlated with each other (r = 0.737, p < 0.01). CONCLUSIONS: Based on the enzyme activity studied, oxidative activity seems to be conserved in the external intercostal muscle of patients with COPD. Activity in the glycolytic pathway seems to increase and the increase is proportional to the severity of COPD. These findings are probably the expression of a combination of adaptive structural factors.

Muscle glycogen stores and meat quality as affected by strategic finishing feeding of slaughter pigs.

The aim of the present study was to investigate whether muscle glycogen stores in slaughter pigs could be decreased through strategic finishing feeding before slaughter. Moreover, preliminary meat quality traits were measured to see whether such a regulation of muscle glycogen stores affected ultimate pH, color, and tenderness in the meat. The strategic finishing feeding was carried out the last 3 wk prior to slaughter. Seven experimental groups with eight animals per group were fed diets low in digestible carbohydrates. A control group with four animals was fed a traditional grower-finishing diet. The muscle glycogen stores were reduced in longissimus muscle (LM) 11 to 26% at the time of slaughter in pigs that were fed the experimental diets compared with the control group. Meat quality measured as ultimate pH and color on LM muscle in half the pigs 24 h postmortem showed that ultimate pH in LM was not affected by the reduction in glycogen stores in the muscles from pigs fed any of the experimental diets. However, the meat from pigs fed the experimental diets was darker than the meat from pigs that were fed the control diet with two of the experimental diets, resulting in significantly lower L* values. Activities of key enzymes in the glycolytic pathway, glycogen phoshorylase a and b, phosphofructokinase, and the fatty acid oxidative pathway, beta-hydrozyacyl-CoA-dehydrogenase, were not affected by the strategic feeding. In contrast, the activity of the proteolytic enzyme calpain as well as its inhibitor calpastatin was influenced by the strategic feeding. Lower activity of mu-calpain and greater activity of calpastatin in the muscle samples from the strategically fed pigs indicate a lesser muscle protein degradation in the muscles compared with muscles of control animals. The present study showed that the muscle glycogen stores in slaughter pigs can be reduced at the time of slaughter through strategic finishing feeding with diets low in digestible carbohydrate without compromising growth rate.

Determination of metabolite and regulatory enzyme levels in Dirofilaria immitis and Ascaris suum: a comparative study.

Studies on metabolite levels in Dirofilaria immitis revealed similarities in several metabolites with those of Ascaris suum. The glycogen level in the filariid was however 3-4 times lower than that in A. suum. Levels of three regulatory enzymes were also determined in D. immitis and compared with those in A. suum. The activities of Hexokinase and Phosphofructokinase were similar. However, the levels of Glycogen phosphorylase b appeared to be much lower in the filariid than in A. suum. The subtle but important differences observed may reflect modifications of the parasite enzymes suggesting salient differences in the regulation of energy production from carbohydrates in the worms. The differences may also represent specialization required for the unique life style of the worms in their different locations in their hosts.

Early and long-term changes of equine skeletal muscle in response to endurance training and detraining.

Twenty-four 4-year-old Andalusian (Spanish breed) stallions were used to examine the plasticity of myosin heavy chain (MHC) phenotype and the metabolic profile in horse skeletal muscle with long-term endurance-exercise training and detraining. Sixteen horses underwent a training programme based on aerobic exercises for 8 months. Afterwards, they were kept in paddocks for 3 months. The remaining eight horses were used as controls. Three gluteus medius muscle biopsy samples were removed at depths of 20, 40 and 60 mm from each horse before (month 0), during (month 3) and after (month 8) training, and again after 3 months of detraining (month 11). MHC composition was analysed by electrophoresis and immunohistochemistry with anti-MHC monoclonal antibodies. Fibre areas, oxidative capacity and capillaries were studied histochemically. The activities of key muscle enzymes of aerobic (citrate synthase and 3-hydroxy-acyl-CoA-dehydrogenase) and anaerobic (phosphofructokinase and lactic dehydrogenase) metabolism and the intramuscular glycogen and triglyceride contents were also biochemically analysed. Early changes with training (3 months) included hypertrophy of type IIA fibres, a reduction of MHC-IIX with a concomitant increase of MHC-IIA, a rise in the number of high-oxidative fibres and in the activities of aerobic muscle enzymes and glycogen content. Long-term changes with training (8 months) were a further decline in the expression of MHC-IIX, an increase of slow MHC-I, additional increases of high-oxidative fibres, capillary density, activities of aerobic enzymes and endogenous glycogen; intramuscular lipid deposits also increased after 8 months of training whereas the activities of anaerobic enzymes declined. Most of exercise-induced alterations reverted after 3 months of detraining. These results indicate that endurance-exercise training induces a reversible transition of MHC composition in equine muscle in the order IIX-->IIA-->I, which is coordinated with changes in the metabolic properties of the muscle. Furthermore, a dose-response relationship was evident between the duration (in total) of training and the magnitude of muscle adaptations.

Infantile phosphofructokinase deficiency with arthrogryposis: clinical benefit of a ketogenic diet.

We report a 2-year-old boy with phosphofructokinase deficiency presenting in the newborn period with congenital arthrogryposis and severe myopathy, who has had significant improvement on a ketogenic diet since its institution at 4 months of age. We provide a rationale for use of this treatment and hypothesize it may be beneficial in other patients with phosphofructokinase deficiency and progressive muscular involvement. Confirmation awaits further clinical trials in carefully selected patients.

A continuous microtiter plate assay for screening nucleotide sugar-synthesizing nucleotidyltransferases.

A continuous microtiter plate nucleotidyltransferase substrate screening assay (NUSSA) is described which allows the identification of nucleotide sugar-synthesizing enzyme activities. The assay is accomplished by the determination of the common product of these enzymes PPi with a PPi-dependent phosphofructokinase. A subsequent enzyme reaction cascade leads to the production of 2 mol NAD per mol PPi. PPi-dependent phosphofructokinase was purified from potato with respect to contaminating enzyme activities which would disturb NUSSA performance. NUSSA allows the quick, simultaneous, and comprehensive check of different sugar 1-phosphate and nucleoside triphosphate substrates using purified pyrophosphorylases or crude extracts of plants, microorganisms, and mammalian tissues. Moreover, NUSSA will assist to evaluate these enzymes for the synthesis of important nucleotide sugars which serve as substrates of glycosyltransferases in carbohydrate syntheses.

Oxidative capacity of the skeletal muscle and lactic acid kinetics during exercise in normal subjects and in patients with COPD.

Early lactic acidosis during exercise and abnormal skeletal muscle function have been reported in chronic obstructive pulmonary disease (COPD) but a possible relationship between these two abnormalities has not been evaluated. The purpose of this study was to compare and correlate the increase in arterial lactic acid (La) during exercise and the oxidative capacity of the skeletal muscle in nine COPD patients (age = 62 +/- 5 yr, mean +/- SD, FEV1 40 +/- 9% of predicted) and in nine normal subjects of similar age (54 +/- 3 yr). Following a transcutaneous biopsy of the vastus laterialis, each subject performed a stepwise exercise test on an ergocycle up to his or her maximal capacity during which 5-breath averages of oxygen consumption (Vo2), and serial La concentration measurements were obtained. From the muscle biopsy specimen, the activity of two oxidative enzymes, citrate synthase (CS) and 3-hydroxyacyl CoA dehydrogenase (HADH), and of three glycolytic enzymes, lactate dehydrogenase, hexokinase, and phosphofructokinase were determined. The La/Vo2 relationship during exercise was fitted by an exponential function in the form La = a + bvo2, where be represents the shape of the relationship. The activity of the oxidative enzymes was significantly lower in COPD than in control subjects (22.8 +/- 3.3 versus 36.8 +/- 8.6 mumol/min/g muscle for CS, and 3.1 +/- 1.1 versus 5.5 +/- 1.4 mumol/min/g for HADH, p < 0.0005) and the increase in lactic acid was steeper in COPD (b = 4.3 +/- 2.0 versus 2.1 +/- 0.2 for normal subjects, p = 0.0005). A significant inverse relationship was found between CS, HADH, and b. No difference was found between the two groups for the glycolytic enzymes. We conclude that in COPD the increase in arterial La during exercise is excessive, the oxidative capacity of the skeletal muscle is reduced, and that these two results are interrelated.

Isozyme analysis of human normal polymorphonuclear leukocyte phosphofructokinase.

Phosphofructokinase (PFK) from human polymorphonuclear leukocytes (PMN) was characterized by immunological titration with subunit specific antibodies, column chromatography on QAE-Sephadex and SDS-polyacrylamide gel electrophoresis. Two different isozymes, M-type and L-type, were found. The M(r) values of the M and L subunits were 79,500 +/- 1,914 and 74,250 +/- 1,258, respectively. The two isozymes presented different kinetic and regulatory properties. The results suggest that PFK from human normal PMN is a mixture of M-type and L-type homotetramers, mainly, with possible minor heterotetrameric forms.

Ergometric and metabolic adaptation to a 5-s sprint training programme.

The effects of 7 weeks of sprint training (repeated 5-s all-out sprints) on maximal power output (Wv,max) determined during a force-velocity test and a 30-s Wingate test (Wpeak) were studied in ten students [22 (SD 2) years] exercising on a cycle ergometer. Before and after training, muscle biopsies were taken from vastus lateralis muscle at rest for the ten subjects and immediately after a training session for five of them. Sprint training induced an improvement both in peak performances by 25% (Wv,max and Wpeak) and in the 30-s total work by 16%. Before sprint training, the velocity reached with no load (v0) was related to the resting muscle phosphocreatine (PCr) stores (r = 0.87, P < 0.001). The training-induced changes in v0 were observed only when these PCr stores were lowest. This pointed to a possible limiting role of low PCr concentrations in the ability to reach a high velocity. The improvement in performances was linked to an increase in the energy production from anaerobic glycolysis. This result was suggested in muscle by the increase in lactate production measured after a training session associated with the 20% higher activity of both phosphofructokinase and lactate dehydrogenase. The sprint training also increased the proportion of slow twitch fibres closely related to the decrease in fast twitch b fibres. This result would appear to demonstrate an appropriate adaptive reaction following high-intensity intermittent training for the slow twitch fibres which exhibit a greater oxidative capacity.

Altered allosteric properties of cytoskeleton-bound phosphofructokinase in muscle from mice with X chromosome-linked muscular dystrophy (mdx).

Intracellular distribution of cytoskeleton-bound and soluble phosphofructokinase (PFK) (the rate-limiting enzyme in glycolysis) in mdx dystrophic muscle was the same as in control nondystrophic muscle. However, the allosteric activity of both bound and soluble PFK was reduced in mdx muscle, accompanied by a decrease in ATP level. In contrast to normal muscle, the cytoskeleton-bound PFK in mdx muscle was sensitive to allosteric regulation, like the soluble enzyme. This change in the properties of cytoskeletal PFK in mdx mice may result from the absence of dystrophin, believed to reside in the cytoskeleton. The findings that cytoskeletal PFK in mdx muscle, although altered, remains bound to cytoskeleton may play a role in muscle structure and function and the mild clinical symptoms in mdx mice.

Solubility behaviour of phosphofructokinase from haemolysate of erythrocytes, reticulocytes and bone marrow cells in poly (ethylene glycol) solutions.

The precipitation profiles of phospofructokinase obtained by addition of poly(ethylene glycol) to haemolysates of erythrocytes, reticulocytes and bone marrow are displaced towards higher polymer concentrations when the pH decreases from 6 to 5 or increases from 6 to 8. In the pH range 5 to 8, the concentration of polymer required to provide any level of precipitation follows the order erythrocytic less than reticulocytic less than bone marrow phosphofructokinase. The precipitation of erythrocytic and reticulocytic phosphofructokinase is enhanced by the presence of F6P and ATP. No effect is observed for bone marrow phosphofructokinase. These results are consistent with an isoenzymatic variation of phosphofructokinase in erythrocytes, reticulocytes and bone marrow cells.

Phosphofructokinase and pyruvate kinase in mouse embryonal carcinoma P19 cells in relation to growth and differentiation.

Two key enzymes of glycolysis, phosphofructokinase and pyruvate kinase, were studied in embryonal carcinoma cells (P19 EC cells) and three differentiated derivatives in relation to growth rate and differentiation state. The growth rates of P19 EC cells and its differentiated derivatives are positively correlated with both the specific activity of phosphofructokinase and the expression of the L-subunit of this enzyme. The specific activity of pyruvate kinase and its isozyme composition is not correlated with growth rate but seems to be correlated with the differentiation state of these cells. The decrease in specific activity of pyruvate kinase during differentiation of P19 EC cells induced by retinoic acid or dimethylsulfoxide preceded the shift from K- to M-type pyruvate kinase. In contrast to aggregates that were treated with dimethylsulfoxide, the specific activity of pyruvate kinase was reduced after aggregation in the presence of retinoic acid. Only after plating dimethylsulfoxide-treated aggregates again in the presence of dimethylsulfoxide, was a decrease in specific activity obtained. Both retinoic acid and dimethylsulfoxide are able to induce a K- to -M shift of pyruvate kinase.

Biochemical characteristics of mammalian diaphragms.

Selected biochemical characteristics of diaphragm muscle were compared among several orders of adult mammals (cattle, swine, rabbit, guinea pig, rat, and mouse) with known differences in resting breathing frequencies (f, range = 15-138). Diaphragms from smaller animals had significantly higher citrate synthase (CS) and phosphofructokinase (PFK) activities and substrate oxidation rates than larger animals. Ranges of activities for CS and PFK were 93-27 and 58-39 mumol.g-1.min-1, respectively; and 34-5 and 19-2 nmol.g-1.min-1 for [U-14C]glucose (GLU) and [1-14C]palmitate (PAL) oxidation, respectively. The percent of native fast myosin (FM) isoforms was significantly different among groups. Mouse diaphragm had the highest % FM (88.6%), whereas the lowest values (7.5%) were observed in cattle diaphragm. Myosin ATPase (M-ATPase, pH 9.8) activity was significantly lower in cattle (0.06 mumol.mg protein-1.min-1) and swine (0.38 mumol.mg protein-1.min-1) diaphragm than in other mammals (range of 1.14-0.67 mumol.mg protein-1.min-1). Correlation coefficients determined among means of measured biochemical parameters and established values of f indicated that CS activity and substrate oxidation rates were significantly correlated with f (r = 0.92, 0.92, 0.86 for CS, GLU, PAL, respectively) and the % FM increased with f. M-ATPase (pH 9.8) was significantly correlated with % FM (r = 0.85), whereas PFK and M-ATPase activities were not closely associated with f. It was concluded that f in mammals is significantly correlated with the biochemical parameters of aerobic capacity and is associated with the percent of FM isoforms in the diaphragm.

Heterogeneity of glycolytic enzyme activity and isozyme composition of pyruvate kinase in breast cancer.

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.

Subunit composition, regulatory properties, and phosphorylation of phosphofructokinase from human gliomas.

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.