Evolution of lymphocytes. Immunoglobulin T of the teleost sea bass (Dicentrarchus labrax): Quantitation of gene expressing and immunoreactive cells.

Immunoglobulin T (IgT) is one of the key effector molecules of jawed vertebrate's adaptive immune system, and in this work we describe the quantitative distribution of IgT-expressing and IgT-producing cells in tissues of the European seabass Dicentrarchus labrax by using mRNA riboprobes and a specific anti-IgT antibody. A polyclonal antiserum (pAb) was prepared by immunizing rabbits with three synthetic peptides deduced from the full length IgT cDNA sequence and located in a surface-exposed CH3 domain of IgT constant region. The obtained antiserum, named RAIgT1, was able to recognize by ELISA immunization antigens and IgT from intestinal mucus and serum. In western blots of head kidney leukocytes lysates the antiserum recognized a 180 kDa polypeptide in non-reducing, and a 75 kDa peptide in reducing conditions. Interestingly, the RAIgT1 pAb crossreacted intensely in western blots with rainbow trout IgT purified from mucus and serum. Antisense mRNA IgT oligonucleotide sequences were employed in in situ hybridization to detect IgT-expressing cells in sections from lymphoid tissues, and positive cells were observed in head kidney, spleen, intestine and gills. By employing RAIgT1 in quantitative immunohistochemistry, the highest number of IgT-producing cells was observed in the gills (9.5 ± 0.7%), followed by intestine (8.4 ± 1.2%), head kidney (6.2 ± 1.4%), and spleen (4.1 ± 0.7%). Interestingly, the number of IgT-B cells showed a regionalization in the intestine, increasing from the proximal to the terminal part. By immunofluorescence and flow cytometry of live leukocytes, the percentages of RAIgT1 stained cells were 34 ± 11% in the intestine, 22 ± 5% in head kidney, 16 ± 7% in spleen, and 9 ± 5% in gills. At the fluorescence microscope, live cells from these tissues showed a typical membrane-associated positivity and a lymphocytic morphology, and no IgT/IgM double positive cells were detected. Immunoreactive cells have been purified from head kidney using magnetic beads, and IgT-enriched cells showed by RT-PCR an enhanced expression of the IgT gene, whereas IgT-depleted cells had an highest expression of IgM and TRß genes. These data describe for the first time a quantitative panel of IgT-expressing and IgT-immunoreactive cells in tissues of a teleost fish species.

Engineered nanoparticles of titanium dioxide (TIO2): Uptake and biological effects in a sea bass cell line.

With the rapid development of nanotechnology there has been a corresponding increase in the application of titanium dioxide nanoparticles (TiO2-NPs) in various consumer and industrial products, consequently their potential health hazards and environmental effects are considered an aspect of great concern. In the present study, in order to assess the impact of TiO2-NPs in the marine environment, the biological effects of TiO2-NPs on a sea bass cell line (DLEC) were investigated. Cells were exposed for 24 h to different concentrations of TiO2-NPs (1, 8, 40, 200 and 1000 µg/ml) or co-exposed with CdCl2 (Cd). The effects of UV light irradiation were also investigated in cells treated with TiO2-NPs and/or Cd. The internalization of TiO2-NPs and the morphological cell modifications induced by the treatments were examined by transmission and scanning electron microscopy, this latter coupled with energy dispersive X-ray spectroscopy (EDS) for particle element detection. In addition, the effects of controlled exposures were studied evaluating the cytotoxicity, the DNA damage and the expression of inflammatory genes. Our study indicates that TiO2-NPs were localized on the cell surface mainly as agglomerates revealed by EDS analysis and that they were uptaken by the cells inducing morphological changes. Photoactivation of TiO2-NPs and/or co-exposure with Cd affects ATP levels and it contributes to induce acute cellular toxicity in DLEC cells dependent on Ti concentration. The inflammatory potential and the DNA damage, this latter displayed through a caspase-3 independent apoptotic process, were also demonstrated. Overall our data suggest that the interaction of TiO2-NPs with marine water contaminants, such as cadmium, and the UV irradiation, may be an additional threat to marine organisms.

Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax.

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 µg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 µg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8)  TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.

Dietary fat differentially modulate the mRNA expression levels of oxidative mitochondrial genes in skeletal muscle of healthy subjects.

BACKGROUND AND AIMS: Different types of dietary fats exert differential effects on glucose and lipid metabolism. Our aim was to evaluate the impact of different dietary fats on the expression of skeletal muscle genes regulating mitochondrial replication and function in healthy subjects. METHODS AND RESULTS: Ten healthy subjects (age 29 ± 3 years; BMI 25.0 ± 3 kg/m(2)) received in a random order a test meal with the same energy content but different composition in macronutrients and quality of fat: Mediterranean (MED) meal, SAFA meal (Lipid 66%, saturated 36%) and MUFA meal (Lipid 63%, monounsaturated 37%). At fast and after 180 min, a fine needle aspiration was performed from the vastus lateralis for determination of mitochondrial gene expression by quantitative PCR. No difference in glucose and triglyceride response was observed between the three meals, while NEFA levels were significantly higher following fat-rich meals compared to MED meal (p < 0.002-0.0001). MED meal was associated with an increased expression, albeit not statistically significant, of some genes regulating both replication and function. Following MUFA meal, a significant increase in the expression of PGC1ß (p = 0.02) and a reduction in the transcription factor PPARδ (p = 0.006) occurred with no change in the expression of COX and GLUT4 genes. In contrast, SAFA meal was associated with a marked reduction in the expression of COX (p < 0.001) PFK (p < 0.003), LPL (p = 0.002) and GLUT4 (p = 0.009) genes. CONCLUSION: Dietary fats differentially modulate gene transcriptional profile since saturated, but not monounsaturated fat, downregulate the expression of genes regulating muscle glucose transport and oxidation.

Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures.

Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A(2A) and D(2) receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D(2)R-CFP or A(2A)R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D(2)R-CFP and A(2A)R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A(2A)R positive MVs were treated with the adenosine A(2A) receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A(2A)Rs were functionally competent in target cells. These findings demonstrate that A(2A) receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

Exercise as a new physiological stimulus for brown adipose tissue activity.

BACKGROUND AND AIM: Brown adipose tissue (BAT) plays a major role in body energy expenditure counteracting obesity and obesity-associated morbidities. BAT activity is sustained by the sympathetic nervous system (SNS). Since a massive activation of the SNS was described during physical activity, we investigated the effect of endurance running training on BAT of young rats to clarify the role of exercise training on the activity and recruitment state of brown cells. METHODS AND RESULTS: Male, 10-week-old Sprague Dawley rats were trained on a motor treadmill (approximately 60% of VO2max), 5 days/week, both for 1 and 6 weeks. The effect of endurance training was valuated using morphological and molecular approaches. Running training affected on the morphology, sympathetic tone and vascularization of BAT, independently of the duration of the stimulus. Functionally, the weak increase in the thermogenesis (no difference in UCP-1), the increased expression of PGC-1α and the membrane localization of MCT-1 suggest a new function of BAT. Visceral fat increased the expression of the FOXC2, 48 h after last training session and some clusters of UCP-1 paucilocular and multilocular adipocytes appeared. CONCLUSION: Exercise seemed a weakly effective stimulus for BAT thermogenesis, but surprisingly, without the supposed metabolically hypoactive effects. The observed browning of the visceral fat, by a supposed white-to-brown transdifferentiation phenomena suggested that exercise could be a new physiological stimulus to counteract obesity by an adrenergic-regulated brown recruitment of adipocytes.

Aspects on the integrative actions of the brain from neural networks to "brain-body medicine".

"Integration" is a key term in describing how nervous system can perform high level functions. A first condition to have "integration" is obviously the presence of efficient "communication processes" among the parts that have to be combined into the harmonious whole. In this respect, two types of communication processes, called wiring transmission (WT) and volume transmission (VT), respectively, were found to play a major role in the nervous system, allowing the exchange of signals not only between neurons, but rather among all cell types present in the central nervous system (CNS). A second fundamental aspect of a communication process is obviously the recognition/decoding process at target level. As far as this point is concerned, increasing evidence emphasizes the importance of supramolecular complexes of receptors (the so called receptor mosaics) generated by direct receptor-receptor interactions. Their assemblage would allow a first integration of the incoming information already at the plasma membrane level. Recently, evidence of two new subtypes of WT and VT has been obtained, namely the tunnelling nanotubes mediated WT and the microvesicle (in particular exosomes) mediated VT allowing the horizontal transfer of bioactive molecules, including receptors, RNAs and micro-RNAs. The physiological and pathological implications of these types of communication have opened up a new field that is largely still unexplored. In fact, likely unsuspected integrative actions of the nervous system could occur. In this context, a holistic approach to the brain-body complex as an indissoluble system has been proposed. Thus, the hypothesis has been introduced on the existence of a brain-body integrative structure formed by the "area postrema/nucleus tractus solitarius" (AP/NTS) and the "anteroventral third ventricle region/basal hypothalamus with the median eminence" (AV3V-BH). These highly interconnected regions operate as specialized interfaces between the brain and the body integrating brain-borne and body-borne neural and humoral signals.

Genomic profiling of carbohydrate metabolism in the ectomycorrhizal fungus Tuber melanosporum.

• Primary carbohydrate metabolism plays a special role related to carbon/nitrogen exchange, as well as metabolic support of fruiting body development, in ectomycorrhizal macrofungi. In this study, we used information retrieved from the recently sequenced Tuber melanosporum genome, together with transcriptome analysis data and targeted validation experiments, to construct the first genome-wide catalogue of the proteins supporting carbohydrate metabolism in a plant-symbiotic ascomycete. • More than 100 genes coding for enzymes of the glycolysis, pentose phosphate, tricarboxylic acid, glyoxylate and methylcitrate pathways, glycogen, trehalose and mannitol metabolism and cell wall precursor were annotated. Transcriptional regulation of these pathways in different stages of the T. melanosporum lifecycle was investigated using whole-genome oligoarray expression data together with real-time reverse transcription-polymerase chain reaction analysis of selected genes. • The most significant results were the identification of methylcitrate cycle genes and of an acid invertase, the first enzyme of this kind to be described in a plant-symbiotic filamentous fungus. • A subset of transcripts coding for trehalose, glyoxylate and methylcitrate enzymes was up-regulated in fruiting bodies, whereas genes involved in mannitol and glycogen metabolism were preferentially expressed in mycelia and ectomycorrhizas, respectively. These data indicate a high degree of lifecycle stage specialization for particular branches of carbohydrate metabolism in T. melanosporum.

C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction.

Micro-vesicles can be released by different cell types and operate as 'safe containers' mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway. In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases.

The effects of human sera conditioned by high-intensity exercise sessions and training on the tumorigenic potential of cancer cells.

PURPOSE: There is growing evidence of an association between physical activity and a reduced risk of cancer and cancer recurrence. The aim of this study was to assess the effects of exercise-conditioned human serum (HS) effects on the proliferative and tumorigenic potential of triple-negative breast cancer (TNBC) and prostate cancer (PC) cells. Moreover, modulated mechanisms and several physiological factors that can predict exercise effects were investigated. METHODS: Thirty healthy sedentary subjects were recruited for the study. The subjects performed two high-intensity endurance cycling (HIEC) sessions before and after a nine-week period of high-intensity interval training (HIIT). Cell tumorigenic capacity affected by HS collected before (t0), immediately after (t1), 4 h (t2), and 24 h (t3) after the HIEC sessions was evaluated by in vitro three-dimensional colony formation. The modulation of molecular pathways was analyzed by western blotting and qPCR in TNBC and PC cells, and in TNBC xenografts in exercised mice. RESULTS: All of the HIEC-conditioned HS (t1, t2, and t3) markedly impacted the proliferative and the microtumor-forming capacity of both TNBC and PC cell lines, while the HS collected from the subjects at rest did not. Modulation of the Hippo and Wnt/ß-catenin pathways by HIEC-conditioned HS before and after the period of HIIT was shown. Multiple linear regression analysis showed relationships between the effects of HIEC-conditioned HS in PC cells, lactate threshold and VO2max. CONCLUSIONS: These results highlight the potential of HIEC bouts in tumor progression control and the importance of optimizing an approach to identify physiological predictors of the effects of acute exercise in tertiary cancer prevention.

Sulfate metabolism in Tuber borchii: characterization of a putative sulfate transporter and the homocysteine synthase genes.

The homocysteine synthase (tbhos) and putative sulfate transporter (tbsul1) genes have been characterized in order to understand the sulfate metabolism and regulation in the ectomycorrhizal fungus Tuber borchii. The analyses of tbsul1 and tbhos nucleotide and deduced amino acid sequences led to the identification of the typical domains shown in homologous proteins. Sulfate starvation condition upregulates both genes. The real-time PCR assay of tbsul1 revealed that gene expression was about threefold higher in mycelia grown under sulfate starvation for 2 days than in the mycelial control and in the same starvation condition, the sulfate uptake increased. Real-time PCR and enzymatic assays showed regulation of tbhos when sulfur sources were lacking, suggesting that a transcriptional regulation of this gene rather than a post-transcriptional one occurred. Furthermore, the tbsul1 and tbhos expression patterns were evaluated during the truffle life cycle, revealing an over-expression in the mature ascomata for both genes. In the ectomycorrhizal tissue, only tbhos was upregulated suggesting its substantial role in T. borchii cysteine synthesis. The regulation of tbsul1 and tbhos occurs primarily at the transcriptional level both during vegetative and fruiting phases and these genes could be directly involved in VOCs production.

Two weekly sessions of combined aerobic and resistance exercise are sufficient to provide beneficial effects in subjects with Type 2 diabetes mellitus and metabolic syndrome.

This study was performed to establish whether only 2 sessions per week of combined aerobic and resistance exercise are enough to reduce glycated hemoglobin (HbA(1c)) and to induce changes in skeletal muscle gene expression in Type 2 diabetes mellitus (DM2) subjects with metabolic syndrome. Eight DM2 subjects underwent a 1-yr exercise program consisting of 2 weekly sessions of 140 min that combined aerobic [at 55-70% of maximal oxygen uptake (VO(2max))] and resistance circuit training [at 60-80% of 1 repetition maximum (RM)]. The training significantly improved VO(2max) (from 33.5+/-3.8 ml/kg/min to 38.2+/-3.5 ml/kg/min, p=0.0085) and muscle strength (p<0.05). Changes over baseline were significant for HbA(1c), reduced by 0.45% (p=0.0084), fasting blood glucose (from 8.8+/-1.5 to 6.9+/-2.2 mmol/l, p=0.0132), waist circumference (from 98.9+/-4.8 to 95.9+/-4.6 cm, p=0.0054), body weight (from 87.5+/-10.7 to 85.7+/-10.1 kg, p=0.0375), systolic blood pressure (from 137+/-15 to 126+/-8 mmHg, p=0.0455), total cholesterol (from 220+/-24 to 184+/-13 mg/dl, p=0.0057), and LDL-cholesterol (from 150+/-16 to 105+/-15 mg/dl, p=0.0004). Mitochondrial DNA/nuclear DNA ratio at 6 and 12 months did not change. There was a significant increase of mRNA of peroxisome proliferator- activated receptor (PPAR)-gamma after 6 months of train - ing (p=0.024); PPARalpha mRNA levels were significantly increased at 6 (p=0.035) and 12 months (p=0.044). The mRNA quantification of other genes measured [mitochondrially encoded cytochrome c oxidase subunit II (MTCO2), cytochrome c oxidase subunit Vb (COX5b), PPARgamma coactivator 1alpha (PGC- 1alpha), glucose transporter 4 (GLUT 4), forkhead transcription factor BOX O1 (FOXO-1), carnitine palmitoyltransferase 1 (CPT-1), lipoprotein lipase (LPL), and insulin receptor substrate 1 (IRS-1)] did not show significant changes at 6 and 12 months. This study suggests that a twice-per-week frequency of exercise is sufficient to improve glucose control and the expression of skeletal muscle PPARgamma and PPARalpha in DM2 subjects with metabolic syndrome.

Characterization and mRNA expression profile of the TbNre1 gene of the ectomycorrhizal fungus Tuber borchii.

This study focuses on the cloning and characterization of the major nitrogen regulator element from the ectomycorrhizal fungus Tuber borchii, TbNre1. Sequence analysis of the predicted protein and complementation experiments in Neurospora crassa demonstrated that the cloned gene is orthologous to areA/nit-2 gene. Transcriptional expression investigations by real-time RT-PCR showed TbNre1 up-regulation in the presence of nitrate or in the absence of nitrogen during free-living mycelium growth. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred nitrogen sources like ammonium and glutamine. Furthermore, TbNre1 mRNA was found to be up-regulated during T. borchii and T. platyphyllos interaction. All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in the free living mycelium and in T. borchii-T. platyphyllos interaction. Finally, the possible role of the transcription factor TBNRE1 in the induction of proteases and virulence-like genes, necessary in ectomycorrhizal establishment, was also discussed.

Combination of Coenzyme Q10 Intake and Moderate Physical Activity Counteracts Mitochondrial Dysfunctions in a SAMP8 Mouse Model.

Aging skeletal muscles are characterized by a progressive decline in muscle mass and muscular strength. Such muscular dysfunctions are usually associated with structural and functional alterations of skeletal muscle mitochondria. The senescence-accelerated mouse-prone 8 (SAMP8) model, characterized by premature aging and high degree of oxidative stress, was used to investigate whether a combined intervention with mild physical exercise and ubiquinol supplementation was able to improve mitochondrial function and preserve skeletal muscle health during aging. 5-month-old SAMP8 mice, in a presarcopenia phase, have been randomly divided into 4 groups (n = 10): untreated controls and mice treated for two months with either physical exercise (0.5 km/h, on a 5% inclination, for 30 min, 5/7 days per week), ubiquinol 10 (500 mg/kg/day), or a combination of exercise and ubiquinol. Two months of physical exercise significantly increased mitochondrial damage in the muscles of exercised mice when compared to controls. On the contrary, ubiquinol and physical exercise combination significantly improved the overall status of the skeletal muscle, preserving mitochondrial ultrastructure and limiting mitochondrial depolarization induced by physical exercise alone. Accordingly, combination treatment while promoting mitochondrial biogenesis lowered autophagy and caspase 3-dependent apoptosis. In conclusion, the present study shows that ubiquinol supplementation counteracts the deleterious effects of physical exercise-derived ROS improving mitochondrial functionality in an oxidative stress model, such as SAMP8 in the presarcopenia phase.

Bdnf expression in rat skeletal muscle after acute or repeated exercise.

Brain derived growth factor (BDNF) gene of rat has a complex structure: at least four 5' untranslated exons regulated by different promoters and one 3' exon containing the encoding region. BDNF is expressed by skeletal muscles in an activity-dependent manner. In this study, BDNF mRNA was analysed by RT-PCR in the soleus muscle following a single (acute) session of running or a training of five days of running (repetitive exercise). Moreover, the expression of the exons was quantitatively analysed by real time RT-PCR. Finally, muscle BDNF protein level was evaluated by western blotting. BDNF mRNA was found to increase over the second day after acute exercise; on the other hand, two peaks (2 and 24 hours after the last session, respectively) in BDNF mRNA level were found after repetitive exercise, but it was similar to that of controls 6 hours after the last session. BDNF protein level progressively increased also after the mRNA went back to the basal level, so suggesting that it cumulates within the cell after acute exercise, whereas it followed the mRNA level time course after repetitive exercise. These results point to the following conclusions: BDNF mRNA is up-regulated by activity, but this response is delayed to the second day after acute exercise; repetitive exercise transiently depresses the expression of BDNF mRNA, so that the over-expression due to the previous day's exercise completely disappears 6 hours after the last exercise session.

Molecular characterisation of a Tuber borchii Smt3 gene.

Tbsmt3 gene from the ectomychorrizal fungus Tuber borchii was identified and sequenced. The Tbsmt3 gene encodes for a protein sharing significant amino acid homology with the yeast SMT3, a ubiquitin-like protein that is post-translationally attached to several proteins involved in many cellular processes. The comparison between the Tbsmt3 genomic and cDNA sequences established that the encoding sequence is interrupted by an intron of 312 bp. Southern blot analysis revealed only one copy of Tbsmt3 gene in the T. borchii genome. Tbsmt3 is expressed in all phases of T. borchii life cycle: mycelium, ectomycorrhiza and ascoma. However, the Tbsmt3 mRNA decreased during fruit body maturation.

The interaction of phosphorylated sugars with human hexokinase I.

Glucose 6-phosphate as well as several other hexose mono- and diphosphates were found by kinetic studies to be competitive inhibitors of human hexokinase I (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) versus MgATP. Limited proteolysis by trypsin does not destroy the hexokinase activity but produces as well-defined peptide map when the digested enzyme is electrophoresed in the presence of sodium dodecyl sulfate. MgATP at subsaturating concentration protects hexokinase from trypsin digestion, while phosphorylated sugars, Mg2+, glucose and inorganic phosphate have no effect. Addition of glucose 6-phosphate to the MgATP-hexokinase complex at a concentration 100-times higher than its Ki was not able to reverse the MgATP-induced conformation of hexokinase, suggesting that the binding of glucose 6-phosphate and MgATP are not mutually exclusive. Similar evidence was also obtained by studies of the induced modifications of ultraviolet spectra of hexokinase by the binding of MgATP, glucose 6-phosphate and both compounds. Among a library of monoclonal antibodies produced against rat brain hexokinase I and that recognize human placenta hexokinase I, one (4A6) was found to be able to modify the Ki of glucose 6-phosphate (from 25 to 140 microM) for human hexokinase I. The same antibody also weakens the inhibition by all the other hexoses phosphate studied without affecting the apparent Km for MgATP (from 0.6 to 0.75 mM) or for glucose. These data support the view for the binding of glucose 6-phosphate at a regulatory site on the enzyme.

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological.

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37 degrees C (pH 7.2) was 10.23 +/- 1.90 mumol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg X ATP2-. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 microM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP4- and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94 +/- 0.02 mumol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92 +/- 0.3 mumol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.

Hexokinase in developing rabbit erythroid cells.

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits separated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Most of the falls in hexokinase activity take place when the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.