Leu-11+ lymphocytes with natural killer (NK) activity are precursors of recombinant interleukin 2 (rIL 2)-induced activated killer (AK) cells

Precursors of activated killer (AK) cells cytotoxic for human noncultured metastatic melanoma and colon carcinoma were characterized. These cells required 3 days incubation with recombinant interleukin 2 (rIL 2) and DNA synthesis for the induction of AK activity. Both negative and positive cell purification methods were used to identify the subpopulation of cells containing AK precursors. By complement-mediated cell depletion studies, AK precursors were largely present in the Leu-11+ fraction, and to a much lesser extent in the Leu-7+ and Leu-2a+ fractions; they were absent in Leu-3a+ and Leu-4+ cells. Lymphocyte subpopulations were then purified with a cell sorter to positively select for the subset containing AK precursors. Leu-11+ cells had the highest level of AK activity and proliferative response when cultured for 3 days with rIL 2 as well as the highest level of NK activity before culture. Leu-7+ cells had neither AK activity nor a proliferative response when cultured with rIL 2, although they still possessed high NK activity. The same levels of AK and NK activity were found in Leu-2a+ and Leu-2a- fractions, but both activities were absent among Leu-4+ and Leu-3a+ cells. Further fractionation with a two-step sorting technique showed that the highest AK activity resided in the Leu-7-Leu-11+ cell fraction. Morphologically, this subfraction was granular lymphocytes. Titration experiments or rIL 2-responsive cells showed that the number of cells required to achieve a comparable level of rIL 2 proliferative response were as follows: 35 X 10(3) cells from unseparated PBL, 10 X 10(3) cells from Leu-11+ cells, 3.3 X 10(3) from Leu-7-Leu-11+ cells, and 640 X 10(3) cells from Leu-7+ cells. These results indicate that the lymphocyte subpopulation that proliferates in the presence of rIL 2 and then develops AK activity was a subpopulation of Leu-11+ granular lymphocytes, which also possessed the highest NK activity. These Leu-11+ cells lacked the antigens defined by the Leu-7, Leu-3a, or Leu-4 antibodies. Although Leu-7+ cells did not respond to rIL 2 by themselves, they may play a role in the induction of AK activity.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

The role of detergent in refolding of GdnHCl-denatured arginine kinase from shrimp Fenneropenaeus Chinensis: the solubilization of aggregate and refolding in detergent solutions.

Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.     

Insulin induces expression of adenosine kinase gene in rat lymphocytes by signaling through the mitogen-activated protein kinase pathway

The activity of adenosine kinase (AK) was significantly impaired in splenocytes isolated from diabetic rats. Administration of insulin to diabetic animals restored AK activity, protein, and mRNA levels in diabetic splenocytes. Experiments performed on cultured rat lymphocytes demonstrated that insulin did not change the stability of AK mRNA. Insulin induced AK gene expression in a dose- and time-dependent manner. Maximal increases in AK mRNA (3.9-fold) and activity level (3.7-fold) were observed at the fourth and fifth hours of cell incubation with 10 nM insulin, respectively. The insulin effect on AK expression was not influenced by dibutyryl cAMP (dcAMP). On the other hand dcAMP weakly increased (1.7-fold) basal expression of AK. Exposure of rat lymphocytes to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or rapamycin, an inhibitor of mTOR, did not affect the ability of insulin to stimulate expression of AK. Prior treatment of the cells with 10 microM PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK) completely blocked insulin-stimulated expression of AK gene. Insulin produced a significant transient increase in the tyrosine phosphorylation of ERK1/2, and PD98059 inhibited this phosphorylation. Furthermore exposure of cells to insulin has resulted in transient phosphorylation of Elk-1 on Ser-383 and sustained elevation of c-Jun and c-Fos protein. The maximal phosphorylation of Elk-1 was observed at 15 min, and was blocked by PD98059. We concluded that insulin stimulates AK gene expression through a series of events occurring sequentially. This includes activation of the MAPK cascade and subsequent phosphorylation of Elk-1 followed by increased expression of c-fos and c-jun genes.     

Human adenylate kinase deficiency associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase

Adenylate kinase deficiency in the erythrocyte is a rare genetic disorder associated with hemolytic anemia. To determine the molecular basis of this disorder, we first cloned the normal gene encoding human cytosolic adenylate kinase (AK1) and determined the structure. The gene was 12 kilobase pairs long and was split into 7 exons. The structures of 5'- and 3'-flanking regions were determined by primer extension and RNA blot analysis. The results showed that two species of mRNA with 0.9 and 2.5 kilobases, which differed at the 3'-end portion, were generated by the AK1 gene. Alu sequences were found in the largest intron (intron 5) and in the noncoding region of exon 7. Next, both alleles of the AK1 gene were cloned from DNA of a patient bearing the adenylate kinase deficiency and their nucleotide sequences determined. A transition (C----T) was found in exon 6 on an allele, which resulted in an Arg to Trp (CGG----TGG) substitution at the 128th residue of AK1. Since chicken AK1 is highly homologous to human AK1 with respect to the amino acid sequence, we introduced an Arg to Trp substitution to chicken AK1 at the same position by oligodeoxynucleotide-directed mutagenesis. The mutant chicken AK1 expressed in Escherichia coli showed a reduced catalytic activity as well as a decreased solubility and a change in affinity to phosphocellulose. Thus it was considered that the observed C----T transition was a cause of the decreased AK1 activity of the patient's erythrocyte. Analysis on phosphocellulose chromatography of erythrocyte AK1 of the patient and parents revealed that the patient's mutant allele was derived from the mother.     

Metabolism of purine nucleosides and phosphoribosylpyrophosphate in thymocytes and splenocytes of various mammalian species

1. Activities of ADA, PNP and AK were measured in splenocytes and thymocytes of newborn children, young horses, pigs, sheep, rats and mice and compared with the activities previously found in peripheral lymphocytes. 2. With all species, except horse, the activity of ADA (per 10(6) cells) was higher in thymocytes than in lymphocytes. Activity of ADA was highest in splenocytes of pig and sheep. Activity of ADA was lowest in all lymphoid cells of the horse and only about 10% of the activity in human splenocytes and lymphocytes. 3. With all species, except horse, the activity of PNP was lower in thymocytes than in lymphocytes. Activity of PNP was highest in human lymphocytes and lowest in ovine thymocytes. 4. Activity of AK is comparable in thymocytes of all species and always lower than the ADA activity. In splenocytes of man, horse and pig the activity of AK is comparable to that in thymocytes. 5. Activity of deoxyguanosine kinase was lowest in rat thymocytes and highest in those of man. 6. When enzyme activities are expressed per milligram of protein, the differences between thymocytes and lymphocytes are less pronounced. 7. Activity of PRPP synthetase per 10(6) cells was comparable in thymocytes, splenocytes and lymphocytes of the same species and between the various species. 8. The concentration of PRPP was lowest in ovine thymocytes and higher in splenocytes than in thymocytes of the same species, except man.     

Synthesis of lipopolysaccharide O side chains by Pseudomonas aeruginosa PAO1 requires the enzyme phosphomannomutase.

We have cloned a lipopolysaccharide (LPS) biosynthetic gene from Pseudomonas aeruginosa PAO1 that complements the defect in the production and incorporation of LPS O side chains in the LPS-rough strain AK1012. This gene was characterized by pulsed-field gel electrophoresis, deletion and restriction mapping of the cloned DNA, and biochemical analysis of the protein product. The cloned DNA was found to map to the 7-to-11-min region of the P. aeruginosa chromosome, and the gene needed for complementation of the LPS-rough phenotype was contained on a 2.6-kb HindIII-SacI fragment. This same size restriction fragment contains the alginate gene algC, which encodes the enzyme phosphomannomutase (PMM) and also maps to this region of the P. aeruginosa chromosome. The LPS-rough strain AK1012 was deficient in PMM activity, and this activity was restored to parental levels when the cloned gene was transferred to strain AK1012. In addition, the cloned gene could complement the PMM deficiency in the algC mutant strain 8858, and the cloned algC gene could restore the LPS-smooth phenotype to strain AK1012. These results indicate that the gene we have cloned is equivalent to the alginate gene algC. We designate this gene pmm to emphasize that it encodes the enzyme PMM, which has been shown to be essential for alginate production, and we demonstrate that PMM activity is required for the LPS-smooth phenotype in P. aeruginosa PAO1.     

Bacterial induction of human activated lymphocyte killing and its inhibition by lipopolysaccharide (LPS)

Peripheral blood lymphocytes were incubated with glutaraldehyde-fixed Salmonella bacteria. This resulted in rapid activation of nonspecific cytotoxic potential of the lymphocytes. Both originally noncytotoxic, high-density Percoll-fractionated cells, and cytotoxic natural killer (NK) cell-enriched low-density cells were activated. The induction of originally noncytotoxic cells into activated killer (AK) cells was apparently independent of interferon (IFN), whereas the activation of the NK cell-enriched fractions also involved IFN production. Neither the AK nor NK activity were associated with significant bactericidal activity. The IFN-independent induction of AK activity was not dependent on the O-antigenic polysaccharide part of the lipopolysaccharide (LPS) on the bacterial cell surface, because both smooth (S) strains with differing O-antigenic structures (S-4,12 and S-6,7) and a rough (Re) strain without O-antigen were effective inducers. Isolated LPS, and especially alkali-hydrolyzed (O-deacylated, detoxified) LPS (ALPS) interfered with the induction of cytotoxicity. At concentrations of 10 to 30 micrograms/ml, ALPS totally inhibited the induction of AK activity without affecting the endogenous NK activity. Thus contact with bacteria can lead to the emergence of AK cells, and a bacterial product can effectively block this activation. These phenomena stress the complexity of interactions with host defenses that can take place during bacterial infection.     

Purine nucleoside metabolizing enzyme activities in mouse thymocytes at different stages of differentiation and maturation

The activities of the enzymes involved in purine nucleoside metabolism, adenosine deaminase (ADA), adenosine kinase (AK), purine nucleoside phosphorylase (PNP) and deoxycytidine kinase (deoxyCRK), were determined in mouse thymocytes at various stages of differentiation and maturation, and compared with those in other tissues. The thymocytes were characterized by high ADA and deoxyCRK activities with high ADA/AK and ADA/PNP ratios and low PNP/deoxyCRK ratio. In fetal thymocytes of 16 gestational days, ADA activity was lower, and PNP, AK and deoxyCRK activities were higher than those in the adult thymocytes. During differentiation of fetal thymocytes, ADA activity increased while PNP and AK activities decreased. DeoxyCRK activity decreased after birth. In spleen T lymphocytes, ADA and deoxyCRK activities were lower and PNP activity was about 2.5-fold higher than in the thymocytes. Thus the differentiation stages of T lymphocytes may be characterized by the absolute levels and the ratios of these enzymes.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

DNA mutation associated with the human butyrylcholinesterase K-variant and its linkage to the atypical variant mutation and other polymorphic sites.

Genomic DNA from two families exhibiting the K-variant phenotype of serum butyrylcholinesterase was amplified by PCR and sequenced to determine the molecular basis of this variant. The K-variant phenotype was found to be associated with a DNA transition from guanine to adenine at nucleotide 1615, which caused an amino acid change from alanine 539 to threonine (GCA----ACA; Ala539----Thr). There was a 30% reduction of serum butyrylcholinesterase activity associated with this mutation. Amplification and sequencing of DNA from a random sample of 47 unrelated people gave a frequency of .128 for the K-variant allele. Thus, 1 person in 63 should be homozygous for the K-variant, making the K-variant the most common butyrylcholinesterase variant. The K-variant mutation was also found to be present in 17 (89%) of 19 butyrylcholinesterase genes containing the point mutation which causes the atypical phenotype of butyrylcholinesterase (GAT----GGT; Asp70----Gly). The presence of the K-variant in the same molecule as the atypical variant does not contribute to the qualitative change in the atypical enzyme, but it most likely accounts for the approximately one-third reduction in Vmax of butyrylcholinesterase activity in atypical serum. Two additional point mutations located in noncoding regions of the gene were also observed to be in linkage disequilibrium with the K-variant mutation. As many as four different point mutations have been identified within a single butyrylcholinesterase gene. Inhibition tests of the enzyme in plasma are usually used to distinguish the K-variant from the usual enzyme when the former is present with the heterozygous atypical variant (AK phenotype vs. UA phenotype). Inhibition tests were performed on plasma enzyme from the four possible genotypic combinations of the heterozygous atypical mutation with or without the K-variant mutation on either allele; we found that the AK phenotype was caused by three genotypes (A/K, AK/K, and U/A) and that the UA phenotype was caused by two genotypes (U/A and U/AK).     

Molecular characterization of recombinant mouse adenosine kinase and evaluation as a target for protein phosphorylation.

The regulation of adenosine kinase (AK) activity has the potential to control intracellular and interstitial adenosine (Ado) concentrations. In an effort to study the role of AK in Ado homeostasis in the central nervous system, two isoforms of the enzyme were cloned from a mouse brain cDNA library. Following overexpression in bacterial cells, the corresponding proteins were purified to homogeneity. Both isoforms were enzymatically active and found to possess K(m) and V(max) values in agreement with kinetic parameters described for other forms of AK. The distribution of AK in discrete brain regions and various peripheral tissues was defined. To investigate the possibility that AK activity is regulated by protein phosphorylation, a panel of protein kinases was screened for ability to phosphorylate recombinant mouse AK. Data from these in vitro phosphorylation studies suggest that AK is most likely not an efficient substrate for PKA, PKG, CaMKII, CK1, CK2, MAPK, Cdk1, or Cdk5. PKC was found to phosphorylate recombinant AK efficiently in vitro. Further analysis revealed, however, that this PKC-dependent phosphorylation occurred at one or more serine residues associated with the N-terminal affinity tag used for protein purification.     

Thymidine kinase and adenosine kinase activities in homogenates of thyroid lobes in hemithyroidectomized rats; effects of melatonin in vitro

OBJECTIVES: Thymidine kinase (TK, EC 2.7.1.21) is a part of the pyrimidine salvage pathway, involved in DNA synthesis. In turn, adenosine kinase (AK, EC 2.7.1.20) functions as a part of the purine metabolic pathway, involved in DNA synthesis. Melatonin (Mel) is an indoleamine which is known to inhibit growth processes in the thyroid gland and also in other endocrine and non-endocrine tissues. The aim of our study was to examine TK and AK activities in homogenates of the rat thyroid lobes remaining after contralateral hemithyroidectomy (hemiTx); additionally, incubations with Mel (10(-6), 10(-9), and 10(-12) M) were performed. METHODS: The experiment was performed on young male Wistar rats (6-week old). The enzyme activities were measured by ascending chromatography and expressed as the amounts of radioactive reaction products of the phosphorylation of dThd (for TK) and of dAdo (for AK). RESULTS: 1. HemiTx increased TK activity in homogenates of the remaining thyroid lobe; 2. Mel increased TK activity in all the groups (intact, sham-operated- and hemiTx-rats), except for the concentrations of 10(-9) and 10(-12) M in the hemiTx-rats, in which the increasing effects of Mel on TK activity reached the borderline statistical significance only; 3. Mel increased the AK activity in intact and in shamTx animals; 4. No statistically significant changes were found in AK activity following Mel in vitro in the incubated remaining thyroid lobes, collected from hemiTx-rats.     

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.     

The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression

Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis vector, and clonal mutants were selected for resistance to the cytotoxic adenosine analog adenine arabinoside (Ara-A). Whereas some Ara-A-resistant clones exhibited disruption of the adenosine kinase (AK) locus, others displayed normal AK activity, suggesting that a second locus had been tagged by the insertional mutagenesis plasmid. These Ara-A(r) AK+ mutants displayed reduced adenosine uptake capability, implying a defect in adenosine transport. Sequences flanking the transgene integration point in one mutant were rescued from a genomic library of Ara-A(r) AK+ DNA, and Southern blot analysis revealed that all Ara-A(r) AK+ mutants were disrupted at the same locus. Probes derived from this locus, designated TgAT, were employed to isolate genomic and cDNA clones from wild-type libraries. Conceptual translation of the TgAT cDNA open reading frame predicts a 462 amino acid protein containing 11 transmembrane domains, a primary structure and membrane topology similar to members of the mammalian equilibrative nucleoside transporter family. Expression of TgAT cRNA in Xenopus laevis oocytes increased adenosine uptake capacity in a saturable manner, with an apparent K(m) value of 114 microM. Uptake was inhibited by various nucleosides, nucleoside analogs, hypoxanthine, guanine, and dipyridamole. The combination of genetic and biochemical studies demonstrates that TgAT is the sole functional adenosine transporter in T. gondii and a rational target for therapeutic intervention.     

Increased oxidative metabolism in phytohemagglutinin-stimulated lymphocytes from patients with systemic lupus erythematosus is associated with serum SSA antibody.

We have examined oxidative metabolism in phytohemagglutinin (PHA)-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE) because increased oxygen free radicals would explain the DNA abnormality previously observed in these cells. Almost no oxidative activity was found in freshly isolated control or lupus lymphocytes or control lymphocytes stimulated with PHA. However, increased oxidative metabolism, measured by nitroblue tetrazolium (NBT) conversion to formazan, was found in PHA-stimulated lymphocytes from 14 of 21 lupus patients. A time course study showed that NBT activity appeared in positive lupus lymphocytes at 1-2 days of PHA stimulation, increased to a maximum at 2-4 days, and diminished thereafter. NBT activity was not related to specific disease symptoms, drug therapy, or serum dsDNA, Sm, RNP, or SSB (La) antibodies. The selected population of lupus patients studied precluded conclusions about NBT activity and disease severity. However, the intensity of NBT response in stimulated lupus lymphocytes was positively correlated with the presence of serum SSA (Ro) antibody. We suggest that increased oxidative activity of SLE lymphocytes generates a chemical change in endogenous DNA in vivo and may be a primary event in the pathogenesis of autoimmunity. Absence of detectable oxidative activity in stimulated lymphocytes in a subgroup of lupus patients suggests that at least two different mechanisms are associated with the altered DNA profiles observed in this disorder.     

Mitochondrial adenylate kinase (AK2) from bovine heart. The complete primary structure

The sequence analysis of adenylate kinase isoenzyme 2 (AK2) was completed using a gas-phase sequencer constructed in our laboratory. The enzyme contains 238 amino acid residues in the following order: (sequence; see text) The four cysteine residues of AK2 were reinvestigated. Cys-41 and Cys-233 contain free thiols, which can be carboxymethylated in the intact protein without loss of enzymic activity. Chemical and model-building studies suggest that the pair Cys-43/Cys-93 forms a disulfide in native AK2. The relative molecular mass of AK2, as deduced from the sequence, is 26104. Other methods, including titration of -SH groups, sedimentation equilibrium ultracentrifugation and gel filtration yielded Mr values in the range from 26 000 to 31 500, each value depending on the respective method of determination. Bovine heart AK2 contains 44 residues more than the homologous isoenzyme AK1 (myokinase). As all but one single insertions and deletions cancel, the higher Mr of AK2 is due to 9 residues preceding the N terminus of AK1, a stretch of 30 residues in the middle of the molecule and 6 residues at the end. AK2 and AK1 are similar in their active-site geometry. In contrast, AK2 does not possess any of the three antigenic sites of AK1, which is consistent with the lack of immunological cross-reactivity between AK1 and AK2.     

非典型腺苷酸激酶AK6/hCINAP的结构和功能

腺苷酸激酶广泛存在于各种生物中,在维持细胞中核苷酸的正常含量以及能量代谢活动中发挥重要作用。腺苷酸激酶6 (AK6,又称人coilin相互作用核ATP酶蛋白hCINAP)是一种非典型的腺苷酸激酶,既具有腺苷酸激酶的活性,又具有ATP酶的活性。本课题组对AK6/hCINAP的结构、酶学特征和生物学功能开展了长期的研究,证明其在调控基因转录、核糖体质量、早期胚胎发育、细胞衰老、细胞代谢、细胞增殖和凋亡、DNA损伤反应、炎症反应、肿瘤发展等诸多生物学过程中均发挥关键作用。本文对AK6/hCINAP的结构特征、生物学功能及上游调控因子进行了总结,阐明该酶生物学作用和分子机制具有重要的生物学意义,有助于开发AK6/hCINAP选择性抑制剂并应用于临床治疗。     

The interaction of recombinant IL-2 with human resting lymphocytes: blocking effects of monoclonal antibodies to IL-2 receptors

Several lines of evidence suggest that subsets of resting lymphocytes naturally express interleukin-2 receptors (IL-2.R). Recombinant IL-2 (rIL-2) induced the enhancement of natural killer (NK) activity, the generation of activated killer (AK) cells, the proliferation of resting lymphocytes, and the production of interferon-gamma (IFN-gamma) in lymphocyte cultures. The subsets of lymphocytes mediating these responses appeared to be heterogeneous, but reside predominantly in nylon wool-passed non-T, non-B cells ("null cells" or T3- cells); in response to rIL-2, only Leu 11+T3- cells showed enhanced NK activity, and both Leu 11+T3- and Leu11-T3- cells showed predominantly AK activity, proliferation and production of IFN-gamma. These findings suggest that the T3- fraction (null cell fraction) contains predominantly cells expressing IL-2.R at the resting state. Unlike the case with activated T cells, however, none of these responses was blocked by any of three monoclonal antibodies to IL-2.R, including anti-Tac antibody at any dilution. These results indicate that IL-2.R on the resting T3- cells possess unique biological features compared to those on activated T or B cells. A most likely explanation is that T3- cells possess higher affinity IL-2.R than activated T or B cells. Other possibilities are also discussed.