Heterologous expression,purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase

The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real M. tuberculosis NAD(+) synthetase. These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.     

Paracrine roles of NAD+ and cyclic ADP-ribose in increasing intracellular calcium and enhancing cell proliferation of 3T3 fibroblasts

CD38 is a bifunctional ectoenzyme synthesizing from NAD(+) (ADP-ribosyl cyclase) and degrading (hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic NAD(+) from isolated murine fibroblasts (Bruzzone, S., Guida, L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular active site of CD38. Here we investigated transwell co-cultures of CD38(+) (transfected) and CD38(-) 3T3 cells in order to establish the role of extracellular NAD(+) and cADPR on [Ca(2+)](i) levels and on proliferation of the CD38(-) target cells. CD38(+), but not CD38(-), feeder cells induced a [Ca(2+)](i) increase in the CD38(-) target cells which was comparable to that observed with extracellular cADPR alone and inhibitable by NAD(+)-glycohydrolase or by the cADPR antagonist 8-NH(2)-cADPR. Addition of recombinant ADP-ribosyl cyclase to the medium of CD38(-) feeders induced sustained [Ca(2+)](i) increases in CD38(-) target cells. Co-culture on CD38(+) feeders enhanced the proliferation of CD38(-) target cells over control values and significantly shortened the S phase of cell cycle. These results demonstrate a paracrine process based on Cx43-mediated release of NAD(+), its CD38-catalyzed conversion to extracellular cADPR, and influx of this nucleotide into responsive cells to increase [Ca(2+)](i) and stimulate cell proliferation.     

Heterogeneity of dendritic cells in the mouse liver: identification and characterization of four distinct populations

Liver dendritic cells (DC) are believed to play important roles in liver immunity, autoimmunity, and in the regulation of hepatic allograft acceptance. However, limited information is available on the phenotypes and functions of DC in the liver. To address this issue, we isolated DC from murine liver using procedures that do not involve collagenase, and characterized the freshly isolated DC population that had not been subjected to in vitro expansion. Thence, based on the expression of CD4, B220, and CD11b, four subsets or groups of hepatic NK1.1(-)CD11c(+) DC were identified with the following phenotypes: B220(+)CD4(+), B220(+)CD4(-), B220(-)CD11b(+), and B220(-)CD11b(-). Each subset was further characterized both phenotypically and functionally. In addition to unique phenotypic expression, each subset displayed different allostimulation capability in mixed lymphocyte reaction assays. All four groups developed DC morphology following in vitro culture with activation agents and synthesized distinct patterns of cytokines in response to different stimuli. Taken together, our results suggest that groups I and II are IFN-alpha-producing plasmacytoid DC, group III cells are myeloid-related DC, while group IV is a heterogeneous population containing both myeloid- and lymphoid-related DC. Our results demonstrate the highly heterogeneous nature of hepatic DC, which is in agreement with the unique requirements for APC in the complex liver environment.     

Molecular basis of the dual functions of 2B4 (CD244)

2B4 belongs to the CD2 family of molecules and is expressed on all NK, gammadelta, and memory CD8(+) (alphabeta) T cells. The murine NK receptor 2B4 exhibits both inhibitory and activating functions, whereas human 2B4 has been reported to be an activating molecule. How murine 2B4 can act both as an activating and inhibitory receptor and what distinguishes its function from human 2B4 have remained largely unknown. We use here a model system that allows the study of human and murine 2B4 under identical and controlled conditions. These studies reveal that both human and mouse 2B4 can activate or inhibit NK cells. We show here that the level of 2B4 expression and the degree of 2B4 cross-linking play a significant role in the regulation of signaling lymphocyte activation molecule-associated protein-mediated activation by 2B4. A high level of 2B4 expression, heavy cross-linking, and relative paucity of signaling lymphocyte activation molecule-associated protein promote inhibitory function. Our studies demonstrate how a single receptor can have opposing function depending on the degree of receptor expression, extent of its ligation, and the relative abundance of certain adaptor molecules. Because the levels of 2B4 and CD48 are dynamically regulated, these findings have implications for the regulation of NK cell function.     

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.     

Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii

A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 mumoles of nicotinamide adenine dinucleotide (NAD(+)) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD(+), deamino-NAD(+), and thio-NAD(+) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP(+)) and thio-NAD(+) with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD(+), pyridinealdehyde AD(+), acetylpyridine deamino AD(+), and pyridinealdehydedeamino AD(+) with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP(+) and thio-NAD(+) was markedly stimulated by 2'-adenosine monophosphate (2'-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD(+) was only slightly affected by phosphate ions and 2'-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD(+) was only slightly influenced by 2'-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD(+) as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD(+) and perhaps a third regulator site for NADP(+) or 2'-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2'-AMP and other nucleotides on the direction of flow of hydrogen between NAD(+) and NADP(+).     

The murine homologue of the T lymphocyte antigen CD28. Molecular cloning and cell surface expression

The human T lymphocyte Ag CD28 (Tp44) is a homodimeric glycoprotein expressed on the surface of a majority of human peripheral T cells and thymocytes. Although exposure of T cells to anti-CD28 mAb does not activate T cells, stimulation of CD28 can synergize with signals transmitted through the TCR or other stimuli to augment proliferation and lymphokine production. We have used a portion of the human CD28 cDNA to isolate a homologous murine cDNA from an EL4 T lymphoma library. The murine clone has 61% nucleotide identity with the human cDNA. Both human and murine sequences exhibit homology with members of the Ig supergene family and CTLA-4, a T cell specific murine gene. Many characteristics of the human CD28 molecule are conserved within the putative murine CD28 polypeptide. The murine cDNA sequence encodes a polypeptide of 218 amino acids that has 68% identity with the human sequence. Both the murine and human molecules are integral membrane glycoproteins with hydrophobic signal peptide sequences and transmembrane region. All five potential N-linked glycosylation sites are conserved and six of the seven cysteine residues of the mouse protein are found in the human CD28 polypeptide. The murine cDNA is encoded by a single copy nonrearranging gene whose expression at the mRNA level is restricted to T cells. A rabbit antiserum was raised against a synthetic peptide corresponding to a hydrophilic portion of the translated murine cDNA sequence. This antiserum identifies an 80-kDa homodimer consisting of disulfide-bonded subunits of 40 kDa that is expressed on splenic T cells, thymocytes, and several T cell tumors, but not on B cells. deglycosylation studies indicate that four of the five N-linked glycosylation sites are used and that the mature core protein has a molecular mass of 25 kDa, close to that predicted by the cDNA sequence. Transfection of the murine cDNA into Chinese hamster ovary cells resulted in the expression of an 80-kDa dimeric molecule that was immunoprecipitated by the antipeptide antiserum. Taken together, these data provide strong support that we have identified the murine homologue of CD28.     

Effects of Aluminum on Immune Functions of Cultured Splenic T and B Lymphocytes in Rats

The effects of Aluminum (Al) exposure on immune functions of cultured splenic T and B lymphocytes of rats were studied. The lymphocytes were isolated from spleen of healthy male Wistar rats weighing 110-120 g. The cultured cells in RPMI-1640 medium were exposed to 0 (control group), 0.035 (low-dose group), 0.07 (medial-dose group), and 0.14 (high-dose group) mg/mL Al(3+) as aluminum trichloride (AlCl(3)) in an incubator under 5% CO(2) at 37°C for 24 h. The T and B lymphocyte proliferation was measured with a tetrazolium dye colorimetric assay. The levels of interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were determined by iodine [(125)I] IL-2, IL-6, and TNF-α radioimmunoassay kits, respectively. The proportions of CD3(+), CD4(+), and CD8(+) T lymphocytes were measured with a flow cytometer. The results showed that the T and B lymphocyte proliferation, the levels of IL-2, IL-6, TNF-α, the proportions of CD3(+) and CD4(+) T lymphocytes, and the ratio of CD4(+)/CD8(+) T lymphocytes were lowered by Al treatments, while the proportion of CD8(+) T lymphocytes was increased. These findings indicate that Al exposure can inhibit the immune functions of splenic T and B lymphocytes of rats in vitro.     

Effects of specific immunotherapy on the B7 family of costimulatory molecules in allergic inflammation

The effect of allergen-specific immunotherapy (IT) on Ag presentation and T lymphocyte stimulation was evaluated by verifying the expression of costimulatory molecules in allergic patients. Thus, CD28 and CTLA-4, B7, and B7-H molecules on immune cells, as well as cytokine production, were analyzed in and out of the pollination period in 30 patients allergic to Betulaceae that had or had nor undergone specific IT. Results showed that IT attenuated the increase in the percentage of CD28(+)CD4 T cells and the decrease in the percentage of CTLA-4(+)CD4(+) T cells seen in untreated individuals. CD19(+)/CD80, CD19(+)/CD86(+), and CD14(+)/CD80(+) APCs were significantly augmented during pollination in unvaccinated individuals. B7-H1-expressing monocytes (CD14(+)) and B lymphocytes (CD19) as well as CD14 and CD19 B7-H1(+)/IL-10(+) APC were augmented in Betulaceae Ag-stimulated cell cultures of vaccinated patients independently of pollination, and were further increased in these individuals during pollination. As a result, the IL-10-IFN-gamma ratio in CD4(+), CD14(+), and CD19(+) cells increased in vaccinated patients, but decreased in unvaccinated individuals during pollination. These data clarify the cellular and molecular basis underlying the recent observation that peripheral expansion of IL-10-producing cells is associated with successful IT. B7-H1 could be an optimal target for IT of allergic diseases using mAbs.     

CD4(+) T cell levels are decreased during active CMV infection in kidney transplant recipients

The numbers of T lymphocytes and T cell subsets (CD2(+), CD3(+), CD4(+), CD8(+)), activated T cells (CD26(+)), B cells (CD19(+)), granulocytes (CD15(+)) and natural killer cells (CD16/56) were monitored by flow cytometry in 79 kidney transplant recipients, 35 of whom had cytomegalovirus infection. The percentages of these cells were correlated with viral load, as determined by cytomegalovirus antigenemia. Development of cytomegaloviral infection coincided with a significant reduction in the percentages of CD4(+) (P < 0.005) and CD3(+) (P < 0.05) cells. Monitoring of lymphocyte subsets may provide useful information on immunological events during cytomegaloviral infection.     

Contribution of exertional hyperthermia to sympathoadrenal-mediated lymphocyte subset redistribution.

The contribution of hyperthermia to the differential leukocytosis of exercise remains obscure. This study examined changes in circulating sympathoadrenal hormone concentrations and patterns of leukocyte and lymphocyte subset (CD3(+), CD4(+), CD8(+), CD19(+), CD3(-)16(+)/56(+)) redistribution during exercise, with and without a significant rise of rectal temperature (T(re)). Ten healthy men [age 26.9 +/- 5.7 (SD) yr, body mass 76.0 +/- 10.9 kg, body fat 13.9 +/- 4.6%, peak O(2) consumption: 48.0 +/- 12.4 ml x kg(-1) x min(-1)] exercised for 40 min (65% peak O(2) consumption) during water immersion at 39 or 18 degrees C. T(re) increased from 37.2 to 39.3 degrees C (P < 0.0001) after 40 min of exercise in 39 degrees C water but was held constant to an increment of 0.5 degrees C during exercise in 18 degrees C water. Application of this thermal clamp reduced exercise-associated increments of plasma epinephrine (Epi) and norepinephrine (NE) by >50% (P < 0.05) and abolished the postexercise increase in cortisol. Thermal clamping also reduced the exercise-induced leukocytosis and lymphocytosis. Multiple regression demonstrated that T(re) had no direct association with lymphocyte subset mobilization but was significantly (P < 0.0001) correlated with hormone levels. Epi was an important determinant of total leukocytes, lymphocytes, and CD3(+), CD4(+), CD8(+), and CD3(-)CD16(+)/56(+) subset redistribution. The relationship between NE and lymphocyte subsets was weaker than that with Epi, with the exception of CD3(-)CD16(+)/56(+) counts, which were positively (P < 0.0001) related to NE. Cortisol was negatively associated with leukocytes, CD14(+) monocytes, and CD19(+) B- and CD4(+) T-cell subsets but was positively related to granulocytes. We conclude that hyperthermia mediates exercise-induced immune cell redistribution to the extent that it causes sympathoadrenal activation, with alterations in circulating Epi, NE, and cortisol.     

Spermine, a natural polyamine, suppresses LFA-1 expression on human lymphocyte

Natural polyamines, spermine, spermidine, and putrescine, play a pivotal role in the regulation of gene expression; therefore, the age-dependent decreases and the disease-dependent increases in polyamine synthesis suggest a possible contribution of polyamines to the age-related and disease-associated changes in cellular function. In this study, we examined the effects of polyamines on the cellular function and the expression of adhesion molecules on human PBMCs from healthy volunteers. Flow cytometry revealed that PBMCs cultured with spermine decreased mean fluorescent intensities (MFIs) of CD11a and CD18 in the lymphocyte light-scattered region, but not in the monocyte region. This suppression was observed in a dose- and time-dependent manner and found nonspecifically on all cell subsets we tested (CD3(+), CD4(+), CD8(+), CD19(+), CD45RA(+), CD45RO(+), CD4(+)CD45RA(+), CD4(+)CD45RO(+), CD8(+)CD45RA(+), CD8(+)CD45RO(+)). The decreases of CD11a and CD18 MFIs were accompanied by the decrease in adherent capacity of PBMCs to HUVECs. Spermine did not hinder cell activities or cell viability. Among 42 healthy volunteers (mean, 49.5 years old; from 26 to 69), blood spermine levels inversely correlated with the CD11a MFIs of cells in the lymphocyte region (r = -0.48; p = 0.001), but not with those in the monocyte region. The effects of spermidine seemed weaker than those of spermine, and blood spermidine levels had no correlation with CD11a MFIs of the lymphocyte region. Putrescine had no effect on the expressions of membrane molecules. Polyamines, especially spermine, decrease LFA-1 (CD11a/CD18) expression on human lymphocyte and adhesion capacity of PBMCs to HUVECs.     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

Selective expansion and partial activation of human NK cells and NK receptor-positive T cells by IL-2 and IL-15.

IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56(+) T cells, but not CD56(-) lymphocytes. All NK and CD56(+) T cell subpopulations tested (CD4(+), CD8(+), CD4(-)CD8(-), alphabetaTCR(+), gammadeltaTCR(+), CD16(+), CD161(+), CD158a(+), CD158b(+), KIR3DL1(+), and CD94(+)) expanded in response to both cytokines, whereas all CD56(-) cell subpopulations did not. Therefore, previously reported IL-15-induced gammadelta and CD8(+) T cell expansions reflect proliferations of NK and CD56(+) T cells that most frequently express these phenotypes. IL-15 also expanded CD8alpha(+)beta(-) and Valpha24Vbeta11 TCR(+) T cells. Both cytokines stimulated cytotoxicity by NK and CD56(+) T cells against K562 targets, but not the production of IFN-gamma, TNF-alpha, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56(+) T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.     

Isolation of a cDNA encoding the human CD38 (T10) molecule, a cell surface glycoprotein with an unusual discontinuous pattern of expression during lymphocyte differentiation

A cDNA clone encoding the human lymphocyte differentiation Ag CD38 was isolated from a mixture of four different lymphocyte CDNA libraries expressed transiently in COS cells and screened by panning with mAb. Transfected COS cells expressed a surface protein of Mr 46,000 that was similar to the native CD38 molecule expressed on the B cell line Daudi and the T cell leukemia HPB-ALL and which was recognized by each of the CD38 specific mAb HIT-2, T16, T168, HB7, 5D2, ICO-18, and ICO-20. The CD38 cDNA sequence predicts an unusual 30-kDa polypeptide with a short N-terminal cytoplasmic tail, and a carboxyl-terminal extracellular domain carrying the four potential N-linked glycosylation sites. The absence of significant homology with other known surface Ag including members of the Ig superfamily ruled out the possibility that CD38 was the human homologue of the murine Qa2 molecule as has been suggested previously. PvuII digests of human genomic DNA revealed a polymorphism linked to the CD38 gene.     

Leptin receptor expression and signaling in lymphocytes: kinetics during lymphocyte activation, role in lymphocyte survival, and response to high fat diet in mice

Leptin has direct effects not only on neuroendocrine function and metabolism, but also on T cell-mediated immunity. We report in this study that leptin receptor (ObR) is expressed on resting normal mouse CD4(+), CD8(+), B cells, and monocyte/macrophages. ObR expression is up-regulated following cell activation, but with different kinetics, in different lymphocyte subsets. Leptin binding to ObR results in increased STAT-3 activation in T cells, with a different activation pattern in resting vs anti-CD3 Ab stimulated T cells. Leptin also promotes lymphocyte survival in vitro by suppressing Fas-mediated apoptosis. B lymphocytes appear to be more susceptible to the antiapoptotic effects of leptin, and they show higher surface expression of ObR, compared with T cells. Moreover, CD4(+) T cells isolated from ObR-deficient mice displayed a reduced proliferative response, compared with normal controls. Furthermore, ObR/STAT-3-mediated signaling in T lymphocytes is decreased in the diet-induced obese mouse model of obesity and leptin resistance. In summary, our findings show that the ObR is expressed on normal mouse lymphocyte subsets, that leptin plays a role in lymphocyte survival, and that leptin alters the ObR/STAT-3-mediated signaling in T cells. Taken together, our data further support the notion that nutritional status acting via leptin-dependent mechanisms may alter the nature and vigor of the immune response.     

Nicotinamide–adenine dinucleotide pyrophosphatase in the growing and aging mosquito

1. The disappearance of pyridine nucleotides during incubation with mosquito homogenates proceeds through the hydrolysis of the pyrophosphate linkage of these compounds as demonstrated by the formation of NMN and AMP from NAD(+). This reaction was also demonstrated by the loss in the coenzyme functioning property of NAD(+) (yeast alcohol dehydrogenase reaction) without a concomitant loss in reactivity towards cyanide. Transglycosidase activity was not observed in the mosquito homogenates, and low concentrations of nicotinamide did not inhibit the NAD(+) splitting activity of these homogenates. These observations are all in accord with the presence in these homogenates of a NAD(+) pyrophosphatase rather than a NADase. 2. The NAD(+) pyrophosphatase is destroyed by boiling, is not heat-activated, and has a pH optimum at pH8.75. In addition to NAD(+), other dinucleotides such as NADP(+), the 3-acetylpyridine and thionicotinamide analogues of NAD(+) and the thionicotinamide analogue of NADP(+), function as substrates in the hydrolysis catalysed by the pyrophosphatase. 3. A decrease in the specific activity of NAD(+) pyrophosphatase was observed during larval development, and a barely detectable activity was found in the pupa and adult. 4. Enzyme activity per organism increased in the larva but decreased to a very low value in the pupa and adult. These results indicate that the decrease in specific activity was due to a decrease in enzyme concentration rather than an increase in amounts of protein.