Lymphocyte Cytosol Research Articles

Second Messengers cAMP, Ca2+, and NO Modulate Functional Properties of Human Lymphocytes under Conditions of Exposure to UV Light

We studied the effect of UV light (240-390 nm) in doses of 75.5-4530 J/m(2) on functional properties and level of intracellular messengers (cAMP, Ca(2+), NO) in lymphocytic cells of the peripheral blood. Correcting effects of UV light in a dose of 151 J/m(2) on cytotoxic activity of lymphocytes against Ehrlich ascetic carcinoma and calcium concentration in lymphocyte cytosol were observed. We found that UV light reduces the cAMP content and increases NO concentration (151-4530 J/m(2)) in photomodified cells. The involvement of adenylate cyclase and phosphoinositide signaling pathway into the mechanisms of modulation of functional properties of lymphocytes after exposure to UV light was demonstrated.

Regulation of phagocyte NADPH oxidase activity: identification of two cytochromeb558activation states

Activation of the phagocyte NADPH oxidase (phox) requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac1/2) with the membrane cytochrome b558, leading to a hemoprotein conformation change. To clarify this mechanism, the phagocyte NADPH oxidase complex was isolated through cytochrome b558 purification after three chromatographic steps. The purified neutrophil complex was constitutively active in the absence of an amphiphile agent with a maximum turnover (125 mol O2(-) x s(-1) x mol heme b(-1)), indicating that cytochrome b558 has been activated by cytosolic proteins and is in an "open conformation," able to transfer a maximum rate of electrons. In contrast, the phox complex prepared with B lymphocyte cytosol shows a lower constitutive turnover (approximately 50 mol O2(-) x s(-1) x mol heme b(-1)). Analysis of phox complex components by Western blot and mass spectrometry showed the presence of cytosolic factors (especially p67-phox) and structural proteins (moesin, ezrin). To investigate the difference in activity of phox complexes, we evaluated the effect of MRP8 and MRP14, specifically expressed in neutrophils, on the activity of the B lymphocyte complex. MRPs induce the switch between the partially and the fully "open" cytochrome b558 conformation. Moreover, their effect was independent of p67-phox. Data point out two potential cytochrome b558 activation states.

Effects of some 1,4-dihydropyridine Ca antagonists on the blast transformation of rat spleen lymphocytes

Ca antagonists of different classes (verapamil, nifedipine, nicardipinc, diltiazem) in a concentration of 10(-5) M and higher are known to suppress Ca2+ transport into the lymphocyte cytosol, changing a normal response of lymphocytes to mitogens and antigens and so inhibiting their proliferation, as well as IL-2-induced cell proliferation, and their receptor expression on the surface of lymphocytes without cell cytotoxicity. In the present work we studied the effect of some 1, 4-dihydropyridines (DHP) such as nimodipine, nicardipine, nifedipine, niludipine, cerebrocrast, etaftoron, as well as metabolites of cerebrocrast: compounds 7 and 8, (four of the last were synthesized in the Latvian Institute of Organic Synthesis) on rat spleen isolated lymphocyte activation and proliferation in vitro following stimulation with the mitogens concanavalin A (Con A) and recombinant interleukin-2 (IL-2), insulin and insulin antibodies. Based on the experimental results we conclude that in low concentrations (10(-7) to 10(-9) M) the tested 1, 4-DHP Ca antagonists stimulated the process of rat spleen lymphocyte proliferation and DNA synthesis, especially cerebrocrast. It is proposed that these Ca antagonists, as well as causing a concentration decrease of Ca2+, also activated phosphodiesterase, which in its turn, suppressed cAMP accumulation in the lymphocytes and eventually increased Ca2+ ion transport in the cells. Cerebrocrast among all the studied DHP Ca antagonists was the most potent in studies of activation of the lymphocytes in the presence of Con A, IL-2 and insulin, which indicates the number of suppressor and helper lymphocytes and formation of insulin and interleukin receptors on their membrane surface. The increase in the lymphocytes suppressive activity produced by this compound effect can prevent diabetes mellitus types I and II at the stages of pre-diabetes, early and distant diabetes, from hyperexpression of insulin and its receptor antibodies.

Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM).

To identify the genes encoding novel immunodominant antigens of Theileria parva a lambda gt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host-sporozoite interaction.

Identification, Isolation, and Characterization of a 32-kDa Fatty Acid-Binding Protein Missing from Lymphocytes in Humans with Bietti Crystalline Dystrophy (BCD)

Bietti crystalline dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by intraretinal lipid inclusions with degeneration of the retina and sclerosis of the choroidal vessels, resulting clinically in progressive night blindness and constriction of the visual fields. Characterization of fatty acid metabolism in Bietti crystalline dystrophy suggested that BCD might result from abnormalities in lipid-binding proteins or one or more enzymes active in fatty acid elongation and desaturation. To further investigate the first possibility, the docosahexaenoic acid-binding proteins (DHABPs) of human lymphocytes from patients with Bietti crystalline dystrophy were studied and compared with those of normal controls. For fatty acid-binding protein (FABP) identification, lymphocyte cytosol was first subjected to Lipidex 1000 chromatography. FABPs were then cross-linked with [14C]22:6n-3 and identified by HPLC and SDS-PAGE. Ten major peaks corresponding to calculated molecular weights of 13, 14, 32, 43, 45, 50, 64, 96, 105, and 186 kDa exhibit high-affinity binding of fatty acids. Significantly, peaks corresponding to two fatty acid-binding proteins of 32 and 45 kDa present in age-matched controls are absent from lymphocytes of patients with BCD. The 32-kDa fatty acid-binding protein present in normal individuals but absent from patients with BCD was isolated from cultured control human lymphocytes, its fatty acid-binding properties were characterized, and its amino acid composition was analyzed. It shows specific binding of 3n-3 fatty acids, consistent with the pattern of abnormalities of lipid metabolism demonstrated in patients with BCD. These results suggest that the 32- and 43-kDa FABPs are reasonable candidates for causing BCD.

Semi-automated radioassay for determination of dihydropyrimidine dehydrogenase (DPD) activity screening cancer patients for DPD deficiency, a condition associated with 5-fluorouracil toxicity

Dihydropyrimidine dehydrogenase (DPD) catalyzes the reduction of the naturally occurring pyrimidines, uracil and thymine, and the fluoropyrimidine anticancer drug, 5-fluorouracil (FUra) to 5,6-dihydropyrimidines. Previous studies have demonstrated that cancer patients who are DPD deficient exhibit severe toxicity (including death) following treatment with FUra. To date, the direct measurement of DPD enzyme activity has been the only reliable method to identify DPD deficient cancer patients. We now report a semi-automated radioassay for measuring DPD activity in human peripheral lymphocytes. Following incubation of lymphocyte cytosol (at a fixed protein concentration of 200 μg) with [6- 14C]FUra at timepoints ranging from 0 to 30 min, samples are ethanol precipitated, filtered and analyzed by HPLC. Determination of radioactivity is accomplished using an in-line flow scintillation analyzer with automatic quantitation of peaks. This method provides the first specific assay for DPD enzyme activity which is rapid, reproducible and sensitive enough to be used in the routine screening of cancer patients for DPD deficiency prior to treatment with FUra.

Characterization of the protein-protein interactions determining the heat shock protein (hsp90.hsp70.hsp56) heterocomplex

We have reported previously that the three heat shock proteins hsp56, hsp70, and hsp90 exist together in a heterocomplex in human lymphocyte cytosol (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). All three of these proteins also exist in the native glucocorticoid receptor heterocomplex isolated from WCL2 cell cytosol and we have recently shown that the three heat shock proteins are present when immunopurified mouse glucocorticoid receptor is reconstituted into a heterocomplex by rabbit reticulocyte lysate (Hutchison, K. A., Scherrer, L. C., Czar, M. J., Ning, Y., Sanchez, E. R., Leach, K. L., Deibel, M. R., Jr., and Pratt, W. B. (1993) Biochemistry 32, 3953-3957). In this work, we show that highly purified mouse hsp90 binds in a reversible equilibrium to immunopurified rabbit hsp56, but hsp56 does not bind to purified mouse hsp70. In contrast to the equilibrium binding of hsp90 to hsp56, purified hsp90 binds poorly or not at all to purified hsp70 unless a third factor from reticulocyte lysate is present to permit complex formation. This hsp70.hsp90 complex-forming factor is heat-labile, and in the presence of this factor and ATP, a heat shock protein heterocomplex can be reconstituted from purified mouse hsp90 and hsp70 and rabbit hsp56 that is present in the factor preparation. Our data are consistent with a model in which hsp56 and hsp70 bind to different sites on hsp90 but do not interact with each other. The presence of hsp56 in the heat shock protein heterocomplex is not stabilized by molybdate but hsp56 is stabilized if the glucocorticoid receptor is present in addition to hsp90 and hsp70.

The O 2− generating oxidase of B lymphocytes: Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulamatous disease

The O 2 − generating NADPH oxidase of human Epstein-Barr virus immortalized B lymphocytes (EBV-B lymphocyes) and the NADPH oxidase of human neutrophils were compared. The capacity of the oxidase of EBV-B lymphocytes to generate O 2 − is 100-fold less than that of neutrophils. Like the oxidase of neutrophils, the oxidase of EBV-B lymphocytes is decreased or abolished in chronic granulomatous disease (CGD). Activation of neutrophil oxidase in an heterologous cell-free system, using human neutrophil membranes and EBV-B lymphocyte cytosol from healthy and CGD patients, combined with immunoblotting investigations of the cytosolic activating factors p47 and p67 involved in O 2 − production, suggests that neutrophils and EBV-B lymphocytes possess similar complements of cytosolic factors p47 and p67. Cytochrome b −245, the major membrane redox component of the O 2 − generating oxidase, is only slightly expressed in the membrane of EBV-B lymphocytes. A sensitive and specific immunocytochemical method for detection of the two subunits of cytochrome b −245 is described; it shows that both subunits are virtually absent in EBV-B lymphocytes from CGD patients deficient in the large subunit.

Protein phosphorylation regulated by cyclic nucleotide-dependent protein kinases in cell extracts and in intact human lymphocytes

A specific 46,000/50,000 molecular weight protein substrate for both cAMP-dependent protein kinase (cAK) and cGMP-dependent protein kinase (cGK) extensively characterized and purified from human 3latelets was found to be present also human T-lymphocytes, B-lymphocytes and other cells and tumour cell lines. This protein termed vasodilator-stimulated phosphoprotein (VASP) was present in cytosol and membranes of lymphocytes. Addition of exogenous purified cAK or cGK to lymphocyte cytosol or membranes converted 80–90% of VASP to its phosphoform. Endogenous VASP phosphorylation in both cytosol and membranes was stimulated by the addition of cAMP but not by cGMP. With intact lymphocytes, prostaglandin E 1 (PGE 1) and prostaglandin E 2 (PGE 2) induced an increase of cAMP converted 70% of VASP to its phosphoform. In contrast, an increase of cGMP was not associated with VASP phosphorylation although cGK was detected in lymphocytes. These data support the hypothesis that VASP phosphorylation may be an important component of cAMP-mediated regulation of lymphocyte function.

Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease.

Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4 beta-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion O2-, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[gamma-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2-, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2- production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.

Diacylglycerol kinase: a key modulator of signal transduction?

Diacylglycerol kinase (DGK) plays a central role in the metabolism of diacylglycerol released as a second messenger in agonist-stimulated cells. The major purified form of the enzyme (80 kDa DGK) is highly abundant in lymphocyte cytosol and may become membrane-associated via phosphorylation by protein kinase C. In addition, there are several kinase subspecies immunologically distinct from the 80 kDa enzyme, which differ markedly in their responses to several compounds such as sphingosine and R59022. Thus, further work on each enzyme species is needed to define the function of DGK in stimulated cells.

Identification of cyclophilin as the erythrocyte ciclosporin-binding protein

Previous studies on the distribution of circulating ciclosporin have shown that the majority of the drug is associated with erythrocytes. In order to investigate the nature of ciclosporin-erythrocyte binding, binding studies were performed on isolated erythrocytes. At therapeutic concentrations (approx. 0.5 μg/ml in whole blood) > 90% of the erythrocyte associated ciclosporin was found in the cytosol The cytosolic binding capacity was approximately (2–2.5) · 10 5 molecules of ciclosporin per cell. A lower affinity binding of the drug to the plasma membrane occurred only at higher ciclosporin concentrations. The ciclosporin-binding species was purified from erythrocyte cytosol using ciclosporin-Affigel affinity chromatography. This revealed a 16 kDa protein, similar in size to the ciclosporin-binding protein, cyclophilin, previously identified in lymphocyte cytosol. Immunochemical analysis using rabbit anti-bovine spleen cyclophilin antisera revealed that the erythrocyte ciclosporin-binding protein was either cyclophilin or a closely related protein. It is concluded that intracellular ciclosporin-binding within erythrocytes is mostly attributable to the presence of a single protein or protein family represented by cyclophilin. The presence of (2–2.5) · 10 5 copies of this binding protein within each erythrocyte is responsible for the ciclosporin found associated with erythrocytes.

Phosphatidylserine and phorbol myristate acetate stimulation of human lymphocyte guanylate cyclase

1. 1. Guanylate cyclase activities of human lymphocyte membrane and cytosol preparations are stimulated up to three-fold by the direct addition of phosphatidylserine. 2. 2. Phorbol 12-myristate 13-acetate (PMA) greatly augments the effect of phosphatidylserine, but has only a small effect when added alone. 3. 3. Stimulation involves an increase in V max, with no change in K m. 4. 4. Inhibitor studies suggest that stimulation may be mediated by protein kinase C, but not by phospholipase or lipoxygenase.

単核球のキニン不活性化作用

Bradykinin-inactivating activity of mononuclear leukocytes was examined using guinea-pig macrophages and lymphocytes. Intact macrophages rapidly inactivated bradykinin. Bradykinin-inactivating activity of macrophages was present in the membrane and cytosol fractions but not in the nuclear and granular fractions. The bradykinin-inactivating activity of the membrane fraction was inhibited by captopril, whereas that of the cytosol fraction was inhibited by N-ethylmaleimide. Bradykinin-inactivating activity of intact macrophages was inhibited by captopril but not by N-ethylmaleimide, suggesting that the membranebound bradykinin-inactivating enzyme rather than the cytosol bradykinin-inactivating enzyme is responsible for the inactivation of bradykinin by intact macrophages. Although intact lymphocytes hardly inactivated bradykinin, lymphocytes contained a bradykinin-inactivating activity in the cytosol fraction but not in the nuclear and particulate fractions. The bradykinin-inactivating enzyme of the lymphocyte cytosol fraction was inhibited by N-ethylmaleimide, and this enzyme was hardly released extracellularly from intact cells during incubation of lymphocytes with bradykinin.

Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages.

Ordinarily packaged in DNA, adenine deoxyribonucleotides are preferentially concentrated in erythrocyte and lymphocyte cytosol in adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. A spectrum of cytosol enzyme activities are defined in terms of reaction velocities, K0.5s, and nucleotide partition after incubation with ribo- and deoxyribonucleotides. AMP and dAMP were dephosphorylated, but only AMP was deaminated in vitro. Although nucleotidase activity is much stronger in lymphocytes, AMP deaminase was the dominant degradative reaction in all erythrocyte and lymphocyte lysates under the conditions specified. For most cytosolic enzymes, ribonucleotides were preferred cofactors, implying that dADP and dATP often may be bystanders at metabolic events. The adenylate kinase-mediated partition of approximately equimolar ribo- and deoxyribonucleotide substrates yielded a very large preponderance of AMP in the monophosphate compartment, the monophosphates alone being directly vulnerable to degradative loss. The adenylate kinase(s) of lymphocytes differed strikingly from those of erythrocytes in reaction velocities with nucleotide cofactors, K0.5s, and in susceptibility to substrate inhibition.

Physico-chemical properties of the hydroxysterol binding protein of human lymphocyte cytosol: Effects of high salt concentrations and molybdate

Side chain-hydroxylated derivatives of cholesterol (OH sterol) inhibiting lymphoblastic transformation bind with high affinity and specificity to a hydroxysterol binding protein (OHSBP) in the cytosol of human lymphocytes. These binding properties of OHSBP suggested some analogies with that of steroid hormone receptors. The observation of a nuclear binding of 25-OH[ 3H]cholesterol prompted us to apply to the cytosolic OH sterol-OHSBP complex the physico-chemical treatments known to ‘activate’ the steroid hormone receptors. A change of sedimentation coefficient from 8.3 to 4.3 S was observed in hypertonic buffer (0.4 M KC1) but the resulting 4.3 S complex dissociates easily whereas the ‘native’ 8.3 S form does not. Moreover, molybdate did not prevent the 8.3→ 4.3 S transformation induced by KCland neither ammonium sulfate precipitation nor increasing temperature had any effect on the sedimentation coefficient of the 8.3 S complex. Thus, several physico-chemical features differentiate the OH sterol-OHSBP complex from steroid hormone receptors. Hydroxysterol Human lymphocyte Specific binding protein Cell division

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 3. Frequent occurrence of genetic variants in some abundant polypeptides of PHA-stimulated peripheral blood lymphocytes

The 100 or so most intensely Coomassie blue-stained polypeptides from PHA-stimulated peripheral blood lymphocytes were analyzed by two-dimensional electrophoresis in combination with family and population studies. Besides polymorphic lymphocyte cytosol 64k polypeptide reported previously, genetic variants were frequently observed in three polypeptides with molecular weights of 100,000, 49,000, and 40,000. All of them occur in the cytosol. These variant polypeptides are charge variants, because they are separated in the isoelectric focusing dimension. It is indicated by family and population studies and cell distribution analysis that the polypeptide with a molecular weight of 100,000 shows a genetic polymorphism determined by two alleles at a new autosomal locus, as described in the following paper. Family and population studies also suggest that a genetic polymorphism defined by alleles at an autosomal locus is present in each of the polypeptides with molecular weights of 49,000 and 40,000. In contrast to the previous reports of the extremely restricted genetic variability of the 100 or so most abundant fibroblast polypeptides, the present data indicate that common genetic variants are present at least in four of the 100 or so most intensely Coomassie blue-stained lymphocyte polypeptides. The result also shows that careful side-by-side comparison of two-dimensional electrophoresis patterns among both parents and their children is an effective method to detect genetic variant polypeptides.

A specific hydroxysterol binding protein in human lymphocyte cytosol

A discriminating system capable of recognizing the oxygenated sterols was investigated in human lymphocytes. After labelling entire cells with 25-hydroxy [3H] cholesterol (10 nM) the cytosol was ultracentrifuged on a linear sucrose density gradient. Bound 25-hydroxy [3H] cholesterol was located in a single peak with a sedimentation coefficient of 8.3 S. Pronase treatment abolished the radioactive peak. This 8.3 S protein had a low binding capacity for 25-hydroxy [3H] cholesterol and probably a high affinity. This last parameter was not determined on account of some difficulties encountered in a cell-free system relating to the physico-chemical properties of 25-hydroxycholesterol. Only the hydroxylated sterols closely related to 25-hydroxycholesterol were capable of specifically binding to the 8.3 S protein, in contrast with cholesterol. This protein differed from the binding proteins of oxygenated derivatives of vitamin D3 and glucocorticoids. With the human lymphocyte as a model and under our experimental conditions, this hydroxylated sterol-binding protein seems to be involved rather in the cell division control than in the regulation of HMG-CoA reductase activity: indeed, the hydroxysterols able to inhibit thymidine [3H] incorporation into DNA are recognized by this protein whereas the hydroxysterols active on HMG-CoA reductase activity without affecting thymidine [3H] incorporation into DNA are not.

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 2. Genetic polymorphism of lymphocyte cytosol 64K polypeptide.

We describe a genetic polymorphism of human lymphocyte cytosol major polypeptide with mol. wt. 64,000, detected in peripheral blood lymphocytes by high resolution two-dimensional electrophoresis. Three different electrophoretic types (1-1, 2-1, 2-2) of the polypeptide have been identified. Family and population studies indicate that the three phenotypes of the polypeptide are determined by two common alleles at a single autosomal locus. The polypeptide occurs in the cytosol and is predominant in peripheral blood lymphocytes, B-lymphoblastoid cells, T-lymphoblastoid cells, lymph node, and spleen. The polypeptide has not been detected HeLa cells, fibroblasts, erythrocytes, serum, and cerebrum. Traces of the polypeptide exist in liver, kidney, and skeletal muscle. It is proposed that the polypeptide and its locus be temporarily designated lymphocyte cytosol 64K polypeptide (LC64K polypeptide) and LC64P, respectively. In a Japanese population, the gene frequencies of LC64P1 and LC64P2 were 0.936 and 0.064, respectively. The data suggest that LC64P is a new locus, product of which shows genetic polymorphism and is associated with the function and/or the structure of lymphocytes.

Mixed function oxidase and glutathione S-transferase activities in normal human peripheral blood lymphocytes

The carcinogenic activity of some chemicals may be related to uncoupling of metabolism mediated by the mixed function oxygenase system. The intermediate formation of reactive epoxides or hydroxylamines may be eliminated by glutathione S-transferase. The ratio between these 2 enzymic activities may indicate if individuals are prone to cancerous development. If adequate cellular samples are available, assay of these enzymes may be used to trace individuals at risk. Therefore the present paper estimates these 2 enzymic activities (i.e., aryl hydrocarbon hydroxylase (AHH) and N-demethylation, the metabolite forming enzyme activities and glutathione S-transferase, the metabolite detoxifying enzymes), in the peripheral blood lymphocytes of 35 subjects. Their median age was 34 years. AHH activity was increased by 52% in the lymphocyte homogenate and by 45% in the microsomal fraction of smokers when compared with non-smokers. The enzyme activity also increased by 73% in the homogenate and by 93% in the microsomes of those taking drugs. The activity of N-demethylase increased by 212% in microsomes of smokers. However, the glutathione S-transferase activity decreased by about 2% in the lymphocyte homogenate and cytosol of smokers. The subjects taking drugs showed a decrease in this enzyme activity by 8% in the homogenate and by 37% in the cytosol.