AK Activity Research Articles

Purification and Characterization of Arginine Kinase from Locust

L-Arginine kinase (AK; ATP:L-arginine N-phosphotransferase; EC 2.7.3.3) catalyzes the reversible transphosphorylation between N-phospho-L-arginine (PArg) and ATP thus buffering cellular ATP levels. AK was purified from the leg muscle of the locust Migratoria manilensis by Sephacryl S-200 HR gel filtration chromatography and DEAE Sepharose CL-6B fast flow anion exchange chromatography to an apparent homogeneity with a recovery of 80%. The enzyme behaved as monomeric protein with molecular mass of about 40 kD, and had a pH and temperature optimum of 8.6 and 30 degrees C, respectively, and a pI of about 6.3. The Michaelis constants for synthesis of PArg are 0.936 and 1.290 mM for L-arginine and ATP, respectively and k(cat)/K(m)(Arg) 174. The activity of AK required divalent cations such as Mg(2+) and Mn(2+). In the presence of Cu(2+) and Zn(2+), AK activity was greatly inhibited. The intrinsic protein fluorescence emission maximum at 330 nm using the excitation wavelength at 295 nm suggested that tryptophan residues are below the surface of the protein and not exposed to solvent.

Comparison between Immunostimulatory Activity and Molecular Structure of Different Polysaccharides

Background: We previously reported that ginsan, a polysaccharide extracted from Panax ginseng had an immunostimulatory activity such as mitogenic activity, activation of macrophages and killer cells, and production of a variety of cytokines which resulted in antitumor and antiseptic effects. We further purified -(16)-glucan and -(26)-fructan from the ginsan with size exclusion and ion-exchange column chromatography successively. In this study, we performed the structure-based activity of ginsan by comparison with known polysacchrides such as -glucan, curdlan, laminarin, levan, dextran, lentinan and OK-432. Methods: To investigate the immunostimulatory activity of several polysaccharide compounds, we investigated the stimulation of lymphocytes proliferation, the generation of activated killer cells and the secretion of nitrites from activated macrophages. Results: Of polysaccharides tested, curdlan and ginsan stimulated lymphocyte proliferation, suggesting that the molecular weight and composition of polysaccharide are dependent on the mitogenic activity. The production of nitric oxide was significantly increased in curdlan, levan, ginsan and its fraction, indicating that fructan has also capacity to activate macrophages and may devote to kill pathogens. In addition, the activation of macrophages was seemed to be independent of molecular weight of polysaccharide. The generation of AK cells was exhibited in order of curdlan, OK-432> F1, ginsan, F3> levan> etc. The AK activity may be dependent on molecular weight and composition of polysaccharides. Conclusion: Unfortunately, purified polysaccharide from ginsan were less active on immunostimulatory activity than mixed compounds of polysaccharides. From the viewpoint of structure and activity relationships, we found several characteristic features.

A Method of Detecting Autolysis of Brewers' Yeast by Measurement of Extracellular Adenylate Kinase Activity

We have developed an assay for monitoring cell leakage and autolysis in yeast slurries. Adenylate kinase (AK; EC 2.7.4.3, ATP:AMP phosphotransferase) catalyses the reaction: 2ADP  ATP + AMP. The ATP produced in this reaction can be measured with high sensitivity and precision by using the firefly luciferase reaction. AK is a constitutively expressed intracellular enzyme, which is not secreted or excreted by healthy yeast cells; therefore, it serves as a useful marker for autolysis. The test is capable of detecting the AK content of single yeast cells and has a dynamic range of more than five decades. Dilution of the sample extends the dynamic range still further. We applied the method to yeast cultures from several breweries in Europe and Africa. We found a correlation between the yeast storage time and temperature and the general physiological condition of the cultures. Healthy yeast cells had low levels of extracellular AK activity (typically <60 units of AK). Older or badly stored cultures released more AK into the surrounding beer. Older cultures could show as high as 500 units of AK; incorrectly stored cultures ranged from 3,000 to 60,000 units. This new method is useful for routine monitoring of yeast leakage and autolysis and should assist breweries seeking to optimize beer foam quality and yeast activity.

Enzymes of lysine metabolism from Coix lacryma-jobi seeds

Lysine, threonine, methionine and isoleucine are synthesized through the aspartate metabolic pathway. The concentrations of soluble lysine and threonine in cereal seeds are very low. Coix lacryma-jobi (coix) is a maize-related grass and the enzymological aspects of the aspartate metabolic pathway are completely unknown. In order to obtain information on lysine metabolism in this plant species, two enzymes involved in the biosynthesis of these amino acids (aspartate kinase 〚AK, EC 2.7.2.4〛 and homoserine dehydrogenase 〚HSDH, EC 1.1.1.3〛) and two enzymes involved in lysine degradation (lysine 2-oxoglutarate reductase 〚LOR, EC 1.5.1.8〛 and saccharopine dehydrogenase 〚SDH, EC 1.5.1.9〛) were isolated and partially characterized in coix seeds. AK activity was inhibited by threonine and lysine separately, suggesting the presence of two isoenzymes, one sensitive to lysine and the other sensitive to threonine, with the latter corresponding to approximately 60% of the total AK activity. In contrast to previous results from other plant species, the threonine-sensitive AK eluted from an ion exchange chromatography column at higher KCl concentration than the lysine-sensitive form. The HSDH activity extracted from the seeds was partially inhibited by threonine, indicating the presence of threonine-sensitive and threonine-resistant isoenzymes. LOR and SDH activities were detected only in the endosperm tissue and co-purified on an anion exchange chromatography column, suggesting that the two activities may be linked on a single bifunctional polypeptide, as observed for other plant species. One single SDH activity band was observed on non-denaturing PAGE gels. The K m for saccharopine of SDH was determined as 0.143 mM and the K m for NAD as 0.531 mM. Although SDH activity was shown to be stable, LOR, AK and HSDH were extremely unstable, under all buffer systems tested.

ADP is produced by firefly luciferase but its synthesis is independent of the light emitting properties

During experiments aimed at understanding the time course of appearance of reaction products in the Photinus pyralis luciferase system, an expected compound with a typical nucleotide UV spectrum was isolated. According to capillary electrophoresis (CE) analysis and 1H, 31P-NMR spectra, it was unambiguously found to be ADP, either with extracted or recombinant enzymes. The ADP synthesis was demonstrated by standard UV spectrophotometric methods and CE analysis. Also, the luciferase produced AMP and ATP from ADP. This reaction was completely inhibited by Ap 5A at 250 nM and was independent of the light emitting properties of the enzyme. The only catalytic mechanism to explain the production of ADP is an intrinsic adenylate kinase activity of luciferase. The K m values of the AK activity are 0.3, 0.7, 0.06 mM for AMP, ADP, and ATP respectively. The multiple enzyme activities of luciferase may be partly responsible for the complex kinetics of light emission by changing the nucleotide concentrations.

Isolation, partial purification and characterization of isoenzymes of aspartate kinase from rice seeds

Summary Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been isolated and partially purified from immature rice (Oriza sativa L.) seeds. Three methods of protein separation (ammonium sulphate precipitation, anion-exchange chromatography and gel filtration chromatography) were used in order to purify and identify the isoenzymes of AK and HSDH. Two peaks of AK activity were eluted from the anion-exchange chromatography column (FFQ-Sepharose) with 230 and 286 mmol/L KCl. The first peak in elution order was inhibited by threonine, while the second peak was strongly inhibited by lysine. An optimum pH of 7.4 was determined for total AK activity. The optimum temperature for AK action was about 35 °C, whereas a combination of 12.5 mmol/L magnesium sulphate and 20 mmol/L ATP produced the highest level of activity. After the ammonium sulphate step, only one form of HSDH that was completely inhibited by threonine, was observed. The threonine-sensitive forms of HSDH and AK co-purified, independent of the protein purification method used exhibiting a molecular mass of 186 kDa. The lysine-sensitive AK had a molecular mass estimated to be 167 kDa. These results confirm the hypothesis that in higher plants, threonine and lysine-sensitive forms of AK are present and suggest that a bifunctional polypetide containing threonine-sensitive AK and HSDH is present is rice seeds.

Lysine Accumulation in Transgenic Tobacco Expressing Dihydrodipicolinate Synthase of Escherichia coli

The dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) gene from E. coli was introduced into tobacco to examine the ability and role of chimeric DHDPS in the biosynthesis of lysine in higher plants. Southern blotting and genetic analysis of six transformed tobacco plants showed that they contained on an average one to three copies. Seeds at T 1 generation of K 11 T-9 showed a 3 resistant: 1 sensitive segregation ratio in the lysine-resistance test with addition of 2 mM lysine. The DHDPS purified from the heterozygous and homozygous K 11 T-9 required ca. 1 mM and ca. 2 mM lysine for 50 % inhibition of enzyme activity, respectively, but only 20 μM lysine in wild type tobacco. The DHDPS activity in the heteroand homozygous K 11 T-9 plant leaves increased ca. 34- and 59-fold as compared with wild type tobacco. The significant increase in the level of free lysine was observed in heterozygous K 11 T-9 (25-fold) and homozygous K 11 T-9 (55-fold) as compared with wild type plants. In transgenic plants, a highly significant correlation was found between the content of free lysine and the level of DHDPS activity. The accumulation levels of lysine were sufficiently high to inhibit the activity of the lysine-sensitive aspartate kinase (AK; EC 2.7.2.4) in vivo , i.e. 49 % and 68 % in heterozygous and homozygous K 11 T-9 plants, respectively, as compared with wild type plants. However, the inhibition of AK activity was not influenced by lysine accumulation in all of the transformed plants, although threonine and methionine contents decreased in homozygous K 11 T-9. The present results showed an important role of the E. coli DHDPS gene in biosynthesis of lysine in higher plants, and introduction of the lysine-insensitive DHDPS gene may confer agronomically useful levels of essential amino acid concentrations in a number of crops and vegetable species.

Genetic alteration of cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunit expression affects calcium currents and beta-endorphin release in AtT-20 clonal pituitary cells.

The role of the cAMP-dependent kinase (AK) in neurotransmission was investigated by genetic alteration of AK subunit expression in AtT-20 clonal pituitary cells. We characterized and compared wild-type [AK(wt)] cells and two clones with different AK activities. The first stably expresses a gene for a mutant AK regulatory subunit (RI) that does not bind cAMP [AK(-)]; the second stably expresses a gene for the catalytic subunit (C) of AK [AK(+)]. Western blot analysis of RI and C subunit expression showed increased expression of both subunits in AK(+) and AK(-) cells relative to AK(wt), with the transfection-induced expression of one subunit producing a compensatory increase in the expression of the other. The basal AK activities varied among the cell types, with AK(+) cells possessing 3-fold higher basal AK activity than AK(wt) cells, and AK(-) cells possessing half the AK activity of AK(wt) cells. Preincubation of cultures with 300 microM 8-(4-chlorophenylthio)-cAMP increased AK activity approximately 4-fold in AK(wt) and AK(+) cells, but was without effect in AK(-) cells. Subsequent addition of 1 microM cAMP in vitro increased AK activity an additional 2- to 3-fold in all cell types. The higher basal AK activity found in AK(wt) and AK(+) cells was associated with larger whole cell calcium currents (approximately 43% and approximately 75% larger than in AK(-) cells, respectively) and faster rates of current rundown. The currents from each cell line had similar voltage-dependent and pharmacological properties, however, and [3H]PN200-110 binding was similar among the cell types. Maximal currents were evoked at clamp potentials of 0-10 mV; currents were inactivated approximately 30% in the steady state at holding potentials of -40 mV compared to -80 mV, and currents were reduced approximately 45% in the presence of nifedipine at -40 mV, but were insensitive to omega-conotoxin GVIA. AK(wt) and AK(+) cells also had higher basal and cAMP-stimulated release of beta-endorphin; control rates were approximately 50% greater, but stimulated rates were approximately 400% greater compared to those in AK(-) cells. We conclude that a greater number of calcium channels were activated by depolarization in the phosphorylated state, that current rundown was largely due to dephosphorylation, and that activation of calcium channels was coupled to the release of beta-endorphin.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize

Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations,Ask LT19 andAsk2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived amino acids in maize. Kernels of the homozygousAsk2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine, compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 μML-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygousAsk and homozygousAsk2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating thatAsk andAsk2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutantAsk2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutantAsk2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor ofAsk2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive inhibition of wildtype AK. Reduced lysine sensitivity of theAsk2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygousAsk2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from theAsk andAsk2 structural genes.

Isolation and properties of lysine-producing mutants with feedback-resistant aspartokinase derived from a Brevibacterium flavum strain with citrate synthase- and pyruvate kinase-defects and feedback-resistant phosphoenolpyruvate carboxylase.

Although the growth of Brevibacterium flavum KH-21 with CSL, PK−, and PCR was resistant to a lysine analogue, AEC, plus Thr at a concentration of 1 to 3 g/l, used conventionally for isolation of AKR mutants, it was inhibited by higher concentrations of them, and recovered partially upon the addition of Lys, DAP and Met. The growth level recovered was equal to that in the presence of Thr alone. Several lysine-producing mutants with AKR were isolated as those resistant to these high concentrations of AEC plus Thr. The representative strain, AH-198, produced 51 g/l of Lys · HCl at maximum, when cultured for 72 hr in a medium containing 100 g/l of glucose, while the parent strain KH-21 produced only 2 g/l. The productivity of strain AH-198 was the same as or a little higher than that of the HD− type lysine-producing strain No. 22 with CSL, PK−, and PCR·I0.5, the concentration of Lys and Thr inhibiting 50% of the AK activity was 40 mm, 267-fold that of the parent enzyme. The Km for Asp and the optimum concentr...

Depressed activities of purine enzymes in lymphocytes of patients infected with human immunodeficiency virus.

Enzyme activities were studied in peripheral blood lymphocytes from patients infected with, or at risk for, infection with human immunodeficiency virus (HIV). No significant differences were observed in the HIV-infected and HIV-seronegative high-risk patients with regard to enzyme activities of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and purine nucleoside phosphorylase (EC 2.4.2.1) in peripheral blood. Adenosine deaminase (EC 3.5.4.4) was significantly (P less than 0.02) depressed in asymptomatic HIV-seropositive patients and HIV-seronegative patients at high risk of HIV infection as compared with a healthy HIV-seronegative population. Adenosine kinase (AK, EC 2.7.1.20) was significantly increased in the asymptomatic seropositive (P less than 0.02) and also in the HIV-seronegative high-risk groups (P = 0.01) compared with the normal controls. AK activity was significantly lower in subjects with AIDS than in the asymptomatic (P less than 0.002) and high-risk groups (P less than 0.01). Taken together, these results indicate that adenosine deaminase and AK activities are influenced by the health of the patient, and that measurement of AK activity may prove useful in monitoring the clinical progress of patients with HIV infection.

Radiation Sensitivity of Resting and Activated Nonspecific Cytotoxic Cells of T Lineage and NK Lineage

Natural killer (NK) cell-mediated killing of tumor cells is a radiation-sensitive function that in most subjects is completely abrogated by treatment of the effector cells with 3,000 cGy. The radiation sensitivity of LAK (lymphokine-activated killer) cells and their precursors, the bulk of which are NK cells, is undetermined. In this study, functional cytotoxicity assays and electron microscopy were used to determine the effect of radiation on the cytotoxic function of NK cells, LAK cells (generated by three-day culture of peripheral blood lymphocytes with IL-2), and LAK cell precursors (lymphocytes irradiated prior to culture with IL-2). For comparison, we analyzed the radiation sensitivity of lectin-dependent cell-mediated cytotoxicity (LDCC), which is primarily a function of CD3+ CD8+ granular lymphocytes. We also analyzed the radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells (AK activity). Following 3,000 cGy irradiation, NK cells retained their ability to bind to tumor cell targets but, as shown by both morphologic and functional analyses, they did not undergo activation after conjugate formation, and were unable to release the content of their granules. In order to evaluate LDCC, lymphocytes were depleted of CD16+ cells and tested in a cytotoxicity assay in the presence of Con A. The radiation sensitivity curve was comparable to that of NK cell-mediated cytotoxicity. IL-2-treated lymphocytes (LAK cells) were relatively radioresistant as compared with untreated NK cells, and their cytotoxic function was not abrogated until treatment with greater than 10,000 cGy. Cells receiving such radiation doses displayed cytoplasmic blebbing and damage of their cytoskeletal structures, with disruption of centrioles and microtubules, and disarray of the intermediate filaments. As was shown with NK cells, irradiated LAK cells formed conjugates with tumor targets but failed to degranulate. The radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells was identical to that of LAK effector cells. Doses up to 2,000 cGy did not prevent generation of LAK cells from blood lymphocytes, but 3,000 cGy did so. Blast transformation similar to that observed in IL-2-stimulated controls occurred when lymphocytes irradiated with 3,000 cGy were cultured with IL-2. These transformed cells were not cytotoxic and displayed a normal cytoskeletal apparatus but did not bear electron-dense granules.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiation sensitivity of resting and activated nonspecific cytotoxic cells of T lineage and NK lineage

Natural killer (NK) cell-mediated killing of tumor cells is a radiation- sensitive function that in most subjects is completely abrogated by treatment of the effector cells with 3,000 cGy. The radiation sensitivity of LAK (lymphokine-activated killer) cells and their precursors, the bulk of which are NK cells, is undetermined. In this study, functional cytotoxicity assays and electron microscopy were used to determine the effect of radiation on the cytotoxic function of NK cells, LAK cells (generated by three-day culture of peripheral blood lymphocytes with IL-2), and LAK cell precursors (lymphocytes irradiated prior to culture with IL-2). For comparison, we analyzed the radiation sensitivity of lectin-dependent cell-mediated cytotoxicity (LDCC), which is primarily a function of CD3+ CD8+ granular lymphocytes. We also analyzed the radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells (AK activity). Following 3,000 cGy irradiation, NK cells retained their ability to bind to tumor cell targets but, as shown by both morphologic and functional analyses, they did not undergo activation after conjugate formation, and were unable to release the content of their granules. In order to evaluate LDCC, lymphocytes were depleted of CD16+ cells and tested in a cytotoxicity assay in the presence of Con A. The radiation sensitivity curve was comparable to that of NK cell-mediated cytotoxicity. IL-2-treated lymphocytes (LAK cells) were relatively radioresistant as compared with untreated NK cells, and their cytotoxic function was not abrogated until treatment with greater than 10,000 cGy. Cells receiving such radiation doses displayed cytoplasmic blebbing and damage of their cytoskeletal structures, with disruption of centrioles and microtubules, and disarray of the intermediate filaments. As was shown with NK cells, irradiated LAK cells formed conjugates with tumor targets but failed to degranulate. The radiation sensitivity of nonspecific cytotoxicity mediated by mitogen-activated T cells was identical to that of LAK effector cells. Doses up to 2,000 cGy did not prevent generation of LAK cells from blood lymphocytes, but 3,000 cGy did so. Blast transformation similar to that observed in IL-2- stimulated controls occurred when lymphocytes irradiated with 3,000 cGy were cultured with IL-2. These transformed cells were not cytotoxic and displayed a normal cytoskeletal apparatus but did not bear electron- dense granules.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution and genetic basis of arginine kinase in wild type and flightless mutants of Drosophila melanogaster

AbstractArginine kinase (AK) activity in Drosophila melanogaster has been localized to specific tissues, traced through developmental stages, and inferred to be the product of a single gene. AK is most abundant in muscle, particularly in larval and adult body‐wall muscle and digestive tract, and in adult indirect flight muscle (IFM) and legs. Low but detectable levels are present in larval imaginal discs, brain, salivary glands, and adult head and reproductive tract. Electrophoretic analysies of a series of developmental stages, as well as prepupal and adult tissues, show only one cytoplasmic isozyme. Since an allozymic variant has the same altered electrophoretic mobility in all stages and tissues, a single structural gene is inferred for AK. A variety of flightless mutants were compared to wild type in regard to AK activity and distribution.

Response of liver and kidney adenylate kinase to fasting and refeeding in three strains of mice

1. 1. The effects of fasting and refeeding on the AK isozymes in liver and kidney were studied in three strains of mice. 2. 2. Our studies showed that changes in total AK activity and AK isozyme patterns were associated with fasting and refeeding. 3. 3. The AK isozyme changes were strain-dependent, differing in kind and degree among the three strains. 4. 4. It was concluded that species, strain and individual isozyme identities should be included in studies defining changes of enzyme activity owing to changes in physiological conditions.

Adenylate kinase: an oncodevelopmental marker in an animal model for human prostatic cancer.

Data are presented demonstrating that adenylate kinase (AK; EC 2.7.4.3) is an oncodevelopmental enzyme in the prostate of Copenhagen rats. We selected the Dunning tumor (dorsal rat prostate) as a model system because it most nearly approximates the human pathology. Four sublines of the tumor (R3327-H, R3327-AT, MAT Lu, and MAT LyLu) were studied. The tumor sublines were maintained as solid tumors in syngeneic rats and as monolayers in tissue culture. AK activity appeared in conjunction with malignant transformation of the dorsal prostate. We also determined the normal developmental enzyme pattern: AK was present in prostates of newborns, but was undetectable in prostates of adults. However, AK increased after castration. Therefore, we propose AK as a potential oncofetal tumor marker in prostatic cancer.

Codominant and recessive 9-β- D-arabinofuranosyladenine-resistant mutation of baby hamster cells

9-β- D-Arabinosyladenine (araA)-resistant mutants of baby hamster kidney (BHK) cells can be classified into 3 classes. In order to gain a better understanding of the mechanism(s) of resistance and the biochemical basis of cytotoxicity of various purine nucleosides, cell hybrids of the mutant and wild-type cells were made and analyzed. The class I araA-resistant, adenosine-kinase-deficient (AK −) allele was shown to be recessive to the wild-type araA-sensitive (AK +) gene. The class II mutant allele, which encodes an altered ribonucleoside diphosphate reductase, was shown to be codominant. The class III mutants show multiple phenotypes, araA r/dAdo r/adenosine sensitive (Ado s) and alteration in AK activity. The araA-and dAdo-resistant alleles of araS10d, ara-16c, and ara-19a in class III mutant/wild-type hybrid cells are all recessive to the wild-type allele, consistent with a common mechanism of resistance. In contrast the Ado s allele of ara-S10d is dominant while those of ara-16c and ara-19a are recessive. The difference may be a reflection of two distinct mechanisms of enhanced Ado sensitivity or, alternatively, it suggests that the sensitivity of the hybrids to Ado is highly dependent on the level of AK activity.

Studies on Cytotoxicity Generated in Human Mixed Lymphocyte Cultures

Abstract In addition to the allospecific killing detected against Con A blasts, 5-day MLC (X vs Ym) cells have a cytotoxic effect against K562 and the autologous (X) lymphoblastoid cell line (auto LCL). This effect is designated as anomalous killing or, AK. Both the AK and allospecific killing could be generated with purified E-rosetting cells, indicating that the effectors were T cells. However, they appeared to belong to different populations, based on three criteria: 1) Upon secondary challenge with the original stimulating cells, allospecific killing displayed a classical memory response. In contrast, peak AK activity against both K562 and the auto LCL was weaker in the secondary, compared to the primary culture. 2) The AK and allo targets did not cross-compete in cold target inhibition tests. 3) AK and allocytotoxicity distributed differently after fractionation of the MLC cells on immobilized immune complexes (IC plates). AK effectors may be similar to natural killer (NK) cells, as they preferentially kill K562 and other NK-sensitive targets. However, the anti-K562 effect was not entirely the result of persisting NK cells because the activity could be generated from Fc receptor-negative, NK-negative precursors. Furthermore, in contrast to NK cells, the AK effector cells could not be efficiently depleted by attachment to IC plates, suggesting that NK and AK differ with respect to expression of Fc receptors. The relationship between these two effects, as well as the conditions involved in activating AK, requires further clarification.

Creatine kinase B-subunit activity in human serum. I. Development of an immunoinhibition method for routine determination of S-creatine kinase B-sununit activity

The properties of an inhibiting antibody directed against the M-subunits of human creatine kinase (EC 2.7.3.2, CK) were investigated in the reaction system recommended by the Scandinavian Committee on Enzymes (S.C.E.). At 37° C the rate of immunoinhibition of human CK M-subunit dependent activity corresponded to a t 1 2 of 38 s giving a k value of −2 per cent · s −1 when samples were incubated in the S.K.E. CK reagent A in the presence of antibody. Under the selected conditions immunoinhibition of S-CK M-subunit activity up to 1800 U/l was 99 per cent completed within 5 minutes using undiluted samples. No inhibition of CK B-subunit activity occurred at the chosen concentration of antibody. The inhibition data were verified using human sera and electrophoretically homogeneous preparations of human CK isoenzymes BB and MM. Sample adenylate kinase (EC 2.7.4.3, AK) was found to constitute a potential source of falsely increased S-CK B activity. As reported in the accompanying paper a S-CK B value of 15 U/l was used as a discrimination value in the diagnosis of acute myocardial infarction. The S.C.E. CK method incorporates a combination of two AK inhibitors adenosine 5'-monophosphate and P 1, P 5-diadenosine 5'-pentaphosphate. However, a frequency analysis of sample AK activities demonstrated that AK activities of more than 8 U/l will occur in about 10 per cent of the cases. Consequently, it was deemed necessary to measure the individual sample blank AK rates. The routine procedure developed thus included three separate measurements: determination of total S-CK and of S-CK B activities in the absence and presence of antibody, respectively. As the immunoinhibition was only 99 per cent complete a value corresponding to one per cent of the total S-CK activity was subtracted from the measured S-CK B activity. Likewise, the individual sample AK activity had to be measured and subtracted from the apparent S-CK B activity. The within-series precision of this S-CK B method at the levels of 10 U/l and 20 U/l corresponded to C.V. values of 10 and 5 per cent, respectively. Day-to-day precision at the level of 417 U/l corresponded to a C.V. of 4 per cent. The day-to-day precision of a control, total CK activity 625 U/l, gave a mean value of 53% ± 3% of CK B activity.

ADENOSINE DEAMINASE /ADA/ AND PURINE METABOLISM IN HUMAN LYMPHOCYTES

Purine metabolism was studied in lymphocytes by assaying adenine- and hypoxanthine phosphoribosyltransferases /APRT and HPRT/, ADA and adenosine kinase /AK/. Also, the incorporation of 14C-adenine, -hypoxanthine and -adenosine into purine compounds was measured in intact cells. The activities of APRT and HPRT were low and correlated with minimal uptake of the 14C-bases. Adenosine was taken up more avidly, followed by conversion into adenine nucleotides by AK and into inosine by ADA. The predominat pathway was usually deamination, shown by 1.3–2.3 times higher ADA than AK activity, and by 1.3–3.4 times higher isotope flow through the pathway. Thus, intracellular adenosine is mainly degraded rather than reutilised, and deficiency of ADA would lead to elevated intracellular levels. Since adenosine is known to be toxic to a number of mammalian cells, this is a possible mechanism for the disturbed lymphocyte function in severe combined immunodeficiency, in which ADA deficiency has been established.