Hexokinase in bone marrow cells in the rabbit

The hexokinase isozymic pattern of circulating reticulocytes fractionated by density gradient ultracentrifugation was studied. All the cellular fractions obtained show similar ratio of hexokinase Ia/hexokinase Ib while a four fold decay in specific activity was evidenced. Bone-marrow cells of anemic rabbits also contain low amounts of HK Ib while this isozymic form is not present in basophilic erythroblasts.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Somatic variation of H-2Kk expression and structure in a T-cell lymphoma: instability, stabilization, high production and structural mutation

In the heterozygous T lymphoma line LDHB, variants which have lost the expression of individual H-2 class I genes are spontaneously generated in vitro at a frequency of 10(-1)-10(-2). A cell line (HK13) in which the class I gene Kk is stably expressed (frequency of loss variants less than 10(-4) was selected from LDHB cells by fluorescence activated cell sorting. Further selection of HK13 cells for high Kk expression led to the isolation of the HK22 line which expresses twice as much Kk as HK13. From HK13 and HK22 cells, spontaneous structural variants of Kk having lost individual serological determinants of the Kk wild-type molecule, were isolated by fluorescence activated cell sorting. Such variants occur at a frequency of 10(-6)-10(-7) per cell per generation. The analysis of these variants indicates that they carry mutations in the Kk structural gene and that HK13 cells express a single Kk gene which appears to be duplicated in HK22 cells. We did not find evidence for the generation of variants expressing 'alien' class I products in the LDHB cell line. The instability of class I gene expression in LDHB cells and the transition to stable expression may represent steps of T-cell differentiation in the thymus.     

An electrophoretic study of the distribution and properties of human hexokinases

An electrophoretic system which gives a clear separation of human hexokinases HK1, HK2 and HK3 is described. The distribution of the hexokinase isozymes in various human tissues, both adult and fetal, is reported. Some properties of the isozymes were investigated. HK2 was found to be more thermolabile than HK1, and there was also a small but significant difference in molecular size. Unlike HK3, HK1 and HK2 are not inhibited by high glucose concentrations. Screening of red cell lysates from 800 unrelated European individuals revealed no genetic variants of HK1 and HK2. However, in view of their difference in properties, it seems probable that the HK1 and HK2 isozymes are determined by separate gene loci.     

Characterization and comparison of chloramphenicol acetyltransferase variants.

1. Variants of chloramphenicol acetyltransferase from a variety of bacterial species have been isolated and purified to homogeneity. They constitute a heterogeneous group of proteins as judged by analytical affinity and hydrophobic ('detergent') chromatography, native and sodium dodecyl sulfate electrophoresis, sensitivity to sulfhydryl specific reagents, steady state kinetic analysis, and reaction with antisera. 2. The most striking observation is that three variants of chloramphenicol acetyltransferase (R factor type III, Streptomyces acrimycini, and Agrobacterium tumefaciens) possess an apparent subunit molecular weight (24,500) which is significantly greater than that of all other variants examined (22,500). The three atypical variants are not identical since they show marked differences in a number of important parameters. 3. Although the fundamental mechanism of catalysis may prove to be identical for all chloramphenicol acetyltransferase variants, there is a wide range of sensitivity to thiol-directed inhibitors among the enzymes studied. 4. Amino acid sequence analysis of the N-termini of selected variants suggests that the qualitative differences among chloramphenicol acetyltransferase variants is a reflection of structural heterogeneity which is most marked in comparisons between variants from Gram-positive and Gram-negative species.     

Differential expression and isozymic composition of sn-glycerol-3-phosphate dehydrogenase in tissues from variant lines of Drosophila melanogaster

The tissue-specific expression and isozymic composition of Drosophila sn-glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8) have been determined for a high-activity control line and two variant lines that alter either the temporal or systemic expression of GPDH through a reduction in rates of polypeptide synthesis. The temporal variant exhibits a reduction in enzyme levels in all larval tissues and in the adult abdomen, while levels of activity in the adult thorax are equal to the control line. Isozymic analyses of these tissues demonstrate that it is the GPDH-3 species that is reduced in a temporal and tissue-specific manner. In contrast, the systemic variant demonstrates a uniform reduction of all isozymic species in each tissue and developmental stage. Analyses of the tissues of F1 hybrid offspring of each variant line and appropriately marked electrophoretic variants demonstrate that the tissue-specific effects observed are due to cis-acting elements that are tightly linked to the structural gene.     

蓖麻蚕不同组织脂酶同工酶的研究

本工作为蚕类同工酶研究中的一部分,研究了蓖麻蚕五龄幼虫不同组织器官酯酶的分布情况,试图逐渐建立酶谱化。目的在于利用聚丙烯酰胺凝胶电泳,检验家蚕的DNA对蓖麻蚕的诱变作用（陈元霖等,1981）,以期供体、受体与转化体之间几种酶谱的异同,从分子生物学的角度对蚕类DNA诱导遗传性变异加以阐述。     

Evidence for the existence of enzymatic variants of beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria.

A comparison of rat brain and liver β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has revealed that significant differences exist between the enzymes with regard to their kinetic and physical properties. In contrast to the liver enzyme, brain β-hydroxybutyrate dehydrogenase is rapidly inactivated at 46° and is unstable when stored at ?20°. The brain dehydrogenase was found to have a larger K_m (apparent) for the 3-acetylpyridine analog of NAD⁺, and a greater energy of activation in the direction of β-hydroxybutyrate oxidation than the liver enzyme. In the reverse direction, the brain and liver dehydrogenase exhibit substrate inhibition by NADH (0.22 mM and 0.36 mM, respectively). The brain and liver β-hydroxybutyrate dehydrogenase did not differ significantly with regard to the Michaelis-Menten constants measured for NAD⁺ and β-hydroxybutyrate. The K_m constants of brain β-hydroxybutyrate dehydrogenase for acetoacetate (0.39 mM) and NADH (0.05 mM) were lower than those determined for the liver enzyme, acetoacetate (0.73 mM) and NADH (0.35 mM) respectively. These results suggest that the β-hydroxybutyrate dehydrogenase from rat brain and liver are isozymic variants.     

Some characteristics of isoenzymic spectrum of hexokinase and glucose levels in hepatomas and liver of the host]

The total activity of hexokinase (HK) and HK isoenzymic spectrum of the normal liver and slowly groming hepatoma 49 did not show any essential differences. However, the HK total activity and the relative and absolute contents of isoenzyme HK-3 were increased in hepatomas 61 and especially in the rapidly growing hepatoma 22-a. The glucokinase activity decreases in the hepatiomas 49 and 61 and disappears in the rapidly growing hepatoma 22-a. The glucose content in hepatoma 49 was slightly lower than in the normal liver, whereas in other hepatoma no traces of glucose could be detected. At low glucose concentration in the medium (0,1 mM), i.e. under conditions simulating those characteristic of tumors in vivo, the predominant form of HK in all hepatomas studied was found to be HK-3. In the liver of hepatoma-bearing mice some shifts in the value of total HK activity and its isoenzymic spectrum, reminding one of those found in hepatomas themselves, were observed. Unequal deviations in the total HK activity and its isoenzymic spectrum in hepatomas with different degrees of malignancy indicate that these characteristics are secondary rather than primary events depending on tumour progression.     

Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the regulation of hepatic pyruvate kinase synthesis in neonatal rats

The L- and M2-type pyruvate kinase from the liver of 1-day old rats demonstrated no significant activation nor inhibition by treatment with cyclic AMP, glucagon or insulin. Neither was there any change in their isozymic composition. By means of incorporation with [3H]leucine followed by immunoprecipitation, the rates of synthesis of both the L- and M2-type pyruvate kinase were not considerably affected by all three modulators. Insulin and glucagon do not direct an immediate change in the synthesis of liver pyruvate kinase and a fluctuation in the insulin/glucagon ratio is not a probable signal for regulating the isozymic expression in the neonatal period.     

草鱼同工酶基因座位多态性的初步研究

采用垂直淀粉凝胶电泳及特异性组织化学染色技术研究了25尾草鱼的6种同工酶系统(LDH,MDH,ADH,GDH,IDH,EST)约18—23个基因座位的遗传变异型。有7个基因座位(s-Mdh-A,Adh—B,Gdh-1,Gdh-2,Est-1,Est-6,Est-14)具有多态性,在其中4个基因座位(s-Mdh—A,Adh-B,Gdh-1,Est-1)上观察到的基因型频率与Hardy-Weinberg定律相符．实验表明草鱼的同工酶基因座位具有明显的多态性。这一结果为草鱼的人工选种和定向育种的可能性提供了依据。对草鱼GDH,EST同工酶的遗传基础、亚基组成以及酶变异的机理等问题进行了讨论。     

Characterization of an anti-Ly-6 monoclonal antibody which defines and activates cytolytic T lymphocytes

HK1.4 mAb was identified based on its ability to stimulate proliferation of cloned murine CTL. Within the lymphoid lineage, mAb HK1.4 bound exclusively to CTL, regardless of the expression of Lyt-2 or MHC restriction. HK1.4 mAb also bound to 40% of bone marrow cells and less than 5% of thymocytes from all mouse strains tested. Based on the tissue distribution of the determinant with which it reacted and the ability to cross-block binding of the anti-Ly-6 mAb H9/25, mAb HK1.4 appeared to react with a product of the Ly-6 locus. However, significant differences were observed between the properties of mAb HK1.4 and other, previously described anti-Ly-6 mAb. Cell proliferation and lymphokine release by cloned CTL were stimulated by culture with mAb HK1.4 alone or in the presence of non-stimulatory levels of IL-2. This proliferation and lymphokine release were not blocked by the addition of soluble anti-Lyt-2 or anti-IL-2R mAb. Activation induced by HK1.4 mAb proceeds in the absence of accessory cells, of cross-linking of the TCR, or the addition of mitogens or PMA. Stimulation of cells by anti-TCR mAb was not blocked by the addition of soluble HK1.4 mAb, and the stimulatory effects of HK1.4 and anti-TCR mAb were not additive. However, IL-2-driven proliferation of CTL clones was dramatically inhibited by the addition of HK1.4 mAb.HK1.4 mAb had no effect on Ag-specific or lectin-facilitated cytolysis. Taken together, these data indicate that mAb HK1.4 operates via an IL-2-independent pathway of activation that is also independent of the TCR.     

党参的体细胞胚发生及不同发育阶段几种同工酶的分析

以党参为材料,在附加0.1 mg·L-1 2,4-D、0.3 mg·L-1 6-BA和3%蔗糖的MS培养基上获得大量发育良好的体细胞胚. 附加6-BA可以使胚性愈伤组织进行芽的分化,而添加0.1%活性炭后仅有根的分化.利用梯度凝胶电泳进行同工酶的研究表明在胚性愈伤组织和体细胞胚之间,酶谱差异比较大;而在不同发育时期的体细胞胚之间,差异较小.并讨论了同工酶酶谱和酶活性变化与体细胞胚发生、发育的关系.     

Pharmacological profiles of the murine gastric and colonic H,K-ATPases

Background

The H,K-ATPase, consisting of α and β subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα₁ and HKα₂ encoded by different genes. The ouabain-resistant gastric HKα₁-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα₂-H,K-ATPase from different species shows poor primary structure conservation of the HKα₂ subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα₁, HKα₂, or HKβ genes.

Methods

Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα₁, HKα₂, or HKβ knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined.

Results

The colonic H,K-ATPase demonstrated slightly greater affinity for K⁺ than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα₂ KO but was observed in HKβ KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα₁-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα₂-H,K-ATPase was not inhibited by these compounds.

Conclusions

These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride.

General Significance

Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.     

Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation

In vitro evidence has suggested a change in the ability of tau bearing mutations associated with fronto-temporal dementia to promote microtubule assembly. We have used a cellular assay to quantitate the effect of both isoform differences and mutations on the physiological function of tau. Whilst all variants of tau bind to microtubules, microtubule extension is reduced in cells transfected with 3-relative to 4-repeat tau. Mutations reduce microtubule extension with the P301L mutation having a greater effect than the V337M mutation. The R406W mutation had a small effect on microtubule extension but, surprisingly, tau with this mutation was less phosphorylated in intact cells than the other variants.     

Comparative aspects of hexokinase and phosphofructokinase activity in intermuscular adipose tissue

1. The gross mass, mean adipocyte volume and activities of hexokinase (HK) and phosphofructokinase (PFK) were measured in adipose tissue from precisely identified intermuscular, superficial and intra-abdominal depots of 56 randomly collected wild and captive mammals and one bird. 2. In all intermuscular depots studied except that medial to the trapezius muscle, the activities of HK and PFK per adipocyte in adipose tissue in the centre of the depot were greater than in superficial and intra-abdominal depots of the same specimen. 3. These data are consistent with the suggestion that intermuscular adipose tissue may act as a local energy supply for adjacent muscles.     

The Futile Cycling of Hexose Phosphates Could Account for the Fact That Hexokinase Exerts a High Control on Glucose Phosphorylation but Not on Glycolytic Rate in Transgenic Potato (Solanum tuberosum) Roots

The metabolism of potato (Solanum tuberosum) roots constitutively over- and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O₂ uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using ¹⁴C-glucose as precursor showed the formation of ¹⁴C-fructose and ¹⁴C-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed.     

Optimal transfection with the HK polymer depends on its degree of branching and the pH of endocytic vesicles

We have recently reported that liposomes in combination with histidine (HK)-containing polymers enhanced the expression of luciferase in transfected cells. In transformed or malignant cell lines, branched HK polymers (combined with liposome carriers) were significantly more effective than the linear HK polymer in stimulating gene expression. In the current study, we found that the linear HK polymer enhanced gene expression in primary cell lines more effectively than the branched polymers. The differences in the optimal carrier (linear versus branched) were not due to initial cellular uptake, size of the complexes or level of gene expression. There was, however, a strong association between the optimal type of HK polymer and the pH of endocytic vesicles (P = 0.0058). By altering the percentage of histidines carrying a positive charge, the endosomal pH of a cell may determine the amount of DNA released from the linear or branched HK polymer. In the two cell lines in which the linear HK was the optimal polymer, the endocytic vesicles were strongly acidic with a pH of <5.0. Conversely, in the four cell lines in which the branched polymers were optimal transfection agents, the pH of endocytic vesicles was >6.0. Furthermore, binding data support the relationship between DNA release from the optimal HK polymer and endosomal pH. The interplay between optimal HK polymers and the endosomal pH may lead to improved gene-delivery polymers tailored to a particular cell.