NAD glycohydrolase activity in dog cardiac tissue.

1. Dog heart tissue suspension hydrolyzes NAD, NADP and NMN, and releases nicotinamide stoichiometrically. 2. Maximum activity was observed at 50 degrees C and the activation energy was 10 kcal/mol. 3. Optimum pH range was 6.2-7.6. 4. Compounds with adenine-ribose moiety increased the enzymatic activity. 5. Nicotinamide released during incubation produced reaction nonlinearity. 6. Km for NAD and NADP were about the same; Vmax was higher for NAD. Similar findings have been reported for rabbit heart. 7. Dog enzyme appears to be more sensitive than the rabbit enzyme to noncompetitive inhibitors. 8. Pyrophosphatase activity was not detected in dog heart in contrast to rabbit and rat heart preparations.     

Methemoglobin reductase variability as related to NAD glycohydrolase activity

NADH-dependent methemoglobin reductase in hemolysates and in partially purified preparations was tested for thermostability and coenzyme dependency. Thermoinactivation of the coenzyme was prevented by NAD 10^?4m, while NADP had no effect.NAD glycohydrolase produced a decrease in methemoglobin reductase activity and a typical change in electrophoretic pattern.No change in molecular weight as detected by electrophoresis on polyacrylamide at various concentrations was found among the multiple bands.     

Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin

The combination of ATP, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), and DTT (dithiothreitol) is known to promote the expression of the NAD glycohydrolase activity of pertussis toxin, which resides in the toxin's S1 subunit. By monitoring changes in electrophoretic mobility, we have found that ATP and CHAPS act by promoting the reduction of the disulfide bond of the S1 subunit. In addition, ATP, CHAPS, and DTT allowed sulfhydryl-alkylating reagents to inactivate the NAD glycohydrolase activity. In the presence of iodo[14C]acetate, the combination of ATP, CHAPS, and DTT increased 14C incorporation into only the S1 subunit of the toxin, indicating that alkylation of this subunit was responsible for the loss of activity. If iodoacetate is used as the alkylating reagent, alkylation can be monitored by an acidic shift in the isoelectric point of the S1 peptide. Including NAD in alkylation reactions promoted the accumulation of a form of the S1 peptide with an isoelectric point intermediate between that of native S1 and that of S1 alkylated in the absence of NAD. This result suggests that NAD interacts with one of the two cysteines of the S1 subunit. In addition, we found the pH optimum for the NAD glycohydrolase activity of pertussis toxin is 8, which may reflect the participation of a cysteine in the catalytic mechanism of the toxin.     

Terminal diols as efficient substrates for transglycosylational activity of NAD glycohydrolase

Linear terminal alkane-diols have been shown to function as more efficient substrates of the transglycosylational activity of NAD+ glycohydrolase (NADase) than the corresponding respective 1-alkanols. A series of eight alkane-diols from ethane-1,2- to nonane-1,9-diols underwent an O-ADP-ribosylation in the incubation reaction with NAD/NADase to provide the corresponding ribosylated products. The structural properties of these products were characterized by 1H NMR and MS spectrometries. No substantial double ADP-ribosylation of the two hydroxy functions was observed which was initially expected in the diols of higher carbon number.     

Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.     

Effect on some pyridine derivatives on the activity of heart NAD glycohydrolase]

Effect of some 3- and 4-substituted pyridines on enzymatic hydrolysis of NAD by rabbit heart muscle NAD-glycohydrolase has been studied. It is shown that some 4-substituted derivatives in contrast with 3-substituted ones produce an inhibitory effect on the enzyme activity. A new efficient inhibitor of rabbit heart muscle NAD-glycohydrolase (I50 = 10(-3) M)--N1-(2-lactyl)-N2-(isonicotinoy)hydrazine, inducing uncompetitive inhibition of hydrolysis of NAD is found. The mechanism of the inhibitory effect of N1-(2-lactyl)-N2-(isonicotinoyl)hydrazine was investigated and the rate equation for enzymatic hydrolysis of NAD in the presence of inhibitor is calculated. It is suggested, that the inhibitory effect of N1-(2-lactyl)-N2-(isonicotinoyl)hydrazine is due to the formation of triple inactive complex inhibitor-enzyme-adenosinediphosphateribose.     

Apparent pyridine nucleotide synthesis in mitochondria: An artifact of NMN and NAD glycohydrolase activity?

Intact and Triton disrupted mitochondria incorporate [¹⁴C]nicotinamide into [¹⁴C]NMN and [¹⁴C]NAD. Dialyzed Triton extracts lose this activity. The ability to form [¹⁴C]NMN is restored by addition of a fraction of boiled mitochondrial extract or of NMN. PRPP and ATP are not required for [¹⁴C]NMN formation. The specific activity of [¹⁴C]NMN formation decreases with serial washing of mitochondria while that of an outer membrane enzyme (kynurenine-3-monooxygenase) remains about constant. These finding suggest that the previously reported synthesis of NMN and NAD by mitochondria may be due to exchange reactions catalyzed by active glycohydrolase(s) in contaminating microsomes.     

Nicotinamide 2-fluoroadenine dinucleotide unmasks the NAD+ glycohydrolase activity of Aplysia californica adenosine 5'-diphosphate ribosyl cyclase

ADP-ribosyl cyclases catalyze the transformation of nicotinamide adenine dinucleotide (NAD+) into the calcium-mobilizing nucleotide second messenger cyclic adenosine diphosphoribose (cADP-ribose) by adenine N1-cyclization onto the C-1' ' position of NAD+. The invertebrate Aplysia californica ADP-ribosyl cyclase is unusual among this family of enzymes by acting exclusively as a cyclase, whereas the other members, such as CD38 and CD157, also act as NAD+ glycohydrolases, following a partitioning kinetic mechanism. To explore the intramolecular cyclization reaction, the novel nicotinamide 2-fluoroadenine dinucleotide (2-fluoro-NAD+) was designed as a sterically very close analogue to the natural substrate NAD+, with only an electronic perturbation at the critical N1 position of the adenine base designed to impede the cyclization reaction. 2-Fluoro-NAD+ was synthesized in high yield via Lewis acid catalyzed activation of the phosphoromorpholidate derivative of 2-fluoroadenosine 5'-monophosphate and coupling with nicotinamide 5'-monophosphate. With 2-fluoro-NAD+ as substrate, A. californica ADP-ribosyl cyclase exhibited exclusively a NAD+ glycohydrolase activity, catalyzing its hydrolytic transformation into 2-fluoro-ADP-ribose, albeit at a rate ca. 100-fold slower than for the cyclization of NAD+ and also, in the presence of methanol, into its methanolysis product beta-1' '-O-methyl 2-fluoro-ADP-ribose with a preference for methanolysis over hydrolysis of ca. 100:1. CD38 likely converted 2-fluoro-NAD+ exclusively into the same product. We conclude that A. californica ADP-ribosyl cyclase can indeed be classified as a multifunctional enzyme that also exhibits a classical NAD+ glycohydrolase function. This alternative pathway that remains, however, kinetically cryptic when using NAD+ as substrate can be unmasked with a dinucleotide analogue whose conversion into the cyclic derivative is blocked. 2-Fluoro-NAD+ is therefore a useful molecular tool allowing dissection of the kinetic scheme for this enzyme.     

Identification of an unusual AT(D)Pase-like activity in multifunctional NAD glycohydrolase from the venom of Agkistrodon acutus

NAD-glycohydrolases (NADases) are ubiquitous enzymes that possess NAD glycohydrolase, ADPR cyclase or cADPR hydrolase activity. All these activities are attributed to the NADase-catalyzed cleavage of C-N glycosyl bond. AA-NADase purified from the venom of Agkistrodon acutus is different from the known NADases, for it consists of two chains linked with disulfide-bond(s) and contains one Cu(2+) ion. Here, we show that AA-NADase is not only able to cleave the C-N glycosyl bond of NAD to produce ADPR and nicotinamide, but also able to cleave the phosphoanhydride linkages of ATP, ADP and AMP-PNP to yield AMP. AA-NADase selectively cleaves the P-O-P bond of ATP, ADP and AMP-PNP without the cleavage of P-O-P bond of NAD. The hydrolysis reactions of NAD, ATP and ADP catalyzed by AA-NADase are mutually competitive. ATP is the excellent substrate for AA-NADase with the highest specificity constant k(cat)/K(m) of 293+/-7mM(-1)s(-1). AA-NADase catalyzes the hydrolysis of ATP to produce AMP with an intermediate ADP. AA-NADase binds with one AMP with high affinity determined by isothermal titration calorimetry (ITC). AMP is an efficient inhibitor against NAD. AA-NADase has so far been identified as the first unique multicatalytic enzyme with both NADase and AT(D)Pase-like activities.     

28,000-dalton polypeptide (p28) of adult T-cell leukemia-associated antigen encoded by 24 S mRNA of human T-cell leukemia virus has an associated protein kinase activity

Human T-cell leukemia virus producer cell line MT-2 was labeled with [32P]phosphoric acid, and its cell extracts were immunoprecipitated with mouse monoclonal antibodies (GIN-7, and KK-1) and rabbit sera (anti-p24, and anti-gp68). Analysis of the immunocomplexes on sodium dodecyl sulfate-polyacrylamide gell electrophoresis revealed that p53, p28, and p19 of adult T-cell leukemia-associated antigens were phosphorylated in vivo. Immunocomplexes of MT-2 cell extract with monoclonal antibody KK-1 were incubated with [gamma-32P]ATP in vitro and it was revealed that the phosphokinase activity was associated with p28. The phosphokinase activity of p28 was specific to the serine residue but was not to the tyrosine residue.     

Mouse lacking NAD+-linked glycerol phosphate dehydrogenase has normal pancreatic beta cell function but abnormal metabolite pattern in skeletal muscle

We surveyed the BALB/cHeA mouse, which lacks cytosolic glycerol phosphate dehydrogenase an enzyme that catalyzes a reaction in the glycerol phosphate shuttle. The other enzyme of this shuttle, mitochondrial glycerol phosphate dehydrogenase, is abundant in skeletal muscle and pancreatic islets suggesting that the shuttle's activity is high in these tissues. Levels of glycerol phosphate (low) and dihydroxyacetone phosphate (high) were very abnormal in nonislet tissue, especially in skeletal muscle. Intermediates situated before the triose phosphates in the glycolysis pathway were increased and those after the triose phosphates were generally low, depending on the tissue. The lactate/pyruvate ratio in muscle was low signifying a low cytosolic NAD/NADH ratio. This suggests that a nonfunctional glycerol phosphate shuttle caused a block in glycolysis at the step catalyzed by glyceraldehyde phosphate dehydrogenase. When exercised, mice were unable to maintain normal ATP levels in skeletal muscle. Blood glucose, serum insulin levels, and pancreatic islet mass were normal. In isolated pancreatic islets insulin release, glucose metabolism and ATP levels were normal, but lactate levels and lactate/pyruvate ratios with a glucose load were slightly abnormal. The BALB/cHeA mouse can maintain NAD/ NADH ratios sufficient to function normally under most conditions, but the redox state is not normal. Glycerol phosphate is apparently formed at a slow rate. Skeletal muscle is severely affected probably because it is dependent on the glycerol phosphate shuttle more than other tissues. It most likely utilizes glycerol phosphate rapidly and, due to the absence of glycerol kinase in muscle, is unable to rapidly form glycerol phosphate from glycerol. Glycerol kinase is also absent in the pancreatic insulin cell, but this cell's function is essentially normal probably because of redundancy of NAD(H) shuttles.     

Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity.

Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.     

Only one of the origin binding forms of SV40 T antigen has helicase activity

SV40 T antigen exists in monomeric and multimeric forms. We have separated the individual components by glycerol gradient centrifugation. Helicase activity is found to be associated with monomeric forms only. Dimers and other multimeric forms have no discernable helicase activity. However, results obtained from DNA binding experiments carried out with separated forms of T antigen indicate that both monomers and dimers bind to region I and region II of SV40 origin of replication. Possibly monomeric T antigen unwinds DNA at the replication fork while both monomeric and dimeric forms are utilized for positioning of T antigen at the origin of replication.     

Mouse submaxillary renin has a protease activity and converts human plasma inactive prorenin to an active form

The activation of inactive prorenin by active renin was investigated. Inactive prorenin extensively purified from human plasma was activated by active renin which had been purified from mouse submaxillary glands by multiple chromatographic steps. The apparent lack of protease activity in renin was puzzling in view of the close similarity of its active site structure with that of acid proteases. After a series of affinity chromatographic steps designed to eliminate minute contaminants, renin was found to contain a very low but finite level of a neutral protease activity which was equivalent to 1/40,000 of that of cathepsin D tested by hemoglobinolytic activity. The protease activity was considered as intrinsic to renin since it co-purified with renin persistently at a constant ratio to the renin activity, was precipitated by a monoclonal antibody specific for renin, showed a neutral pH optimum of the enzyme activity in the same pH range as that of renin, and was inhibited by pepstatin. The neutral protease activity is likely to mediate the activation of inactive prorenin.     

CD45 tyrosine phosphatase activity and membrane anchoring are required for T-cell antigen receptor signaling.

T cells that lack the CD45 transmembrane tyrosine phosphatase have a variety of T-cell receptor (TCR) signaling defects that are corrected by reexpression of wild-type CD45 or its intracytoplasmic domains. In this study, a chimeric molecule containing the myristylation sequence of Src and the intracellular portion of CD45, previously shown to restore function in CD45- T cells, was mutagenized to determine if membrane-associated CD45 tyrosine phosphatase activity is required to restore TCR-mediated signaling in CD45- T cells. Abolition of enzymatic activity by substitution of a serine for a critical cysteine in the first catalytic domain resulted in failure of this molecule to restore TCR signaling. Another mutation, in which a single amino acid substitution destroyed the myristylation site, resulted in failure of the chimeric molecule to partition to the plasma membrane. Although expressed at high levels and enzymatically active, this form of intracellular CD45 also failed to restore normal signaling in CD45- T cells. These findings strongly suggest that CD45's function in TCR signaling requires its proximity to membrane-associated tyrosine phosphatase substrates.     

Mouse virulent strain of Staphylococcus epidermidis. Relation of antiphagocytic activity to the protection-inducing antigen.

Using 10(9) or 10(7) colony-forming units of a strain of Staphylococcus epidermidis (strain 1142) in saline or 5% mucin, respectively, 90 to 100% of mice died within 24 to 48 hr after intraperitoneal challenge infection. These organisms gradually multiplied in the peritoneal cavity when injected intraperitoneally into mice, while the mouse avirulent strain (strain 1124) rapidly decreased and no organisms were found there 20 hr after injection. This strain was capable of inducing resistance against challenge with homologous strains. The resistance appeared as early as the first week and disappeared the 4th week after the immunization. However, no resistance was induced with strain 1124 against challenge with strain 1142. Also, hyperimmune rabbit serum prepared with strain 1142 passively protected against challenge with homologous strain in mice. The protective antibody was absorbed out with homologous organisms but not with strain 1124. Subsequently, a surface substance was obtained from strains 1142 or 1124 by the method of Morse. The 1142 surface substance was capable of inducing a resistance against challenge with the homologous strain but not with the 1124 surface substance. Also, this substance absorbed the protective antibody in hyperimmune rabbit serum prepared with the homologous strain but not with the 1124 surface substance nor with the Smith surface antigen extracted from the Smith strain of Staphylococcus aureus. Conversely, the protective antibody in rabbit anti-Smith strain serum against challenge with the homologous strain was absorbed with the Smith surface antigen but not with the 1142 surface substance. In the agar diffusion test, the 1142 surface substance and the Smith surface antigen produced single precipitin lines only against homologous antisera. Biochemical analysis of the 1142 surface substance showed that the substance contained neither nucleic acids nor proteins but is composed of hexosamine, glycerol, phosphorus, alanine, glycine and phenylalanine.     

Conditional control of chimeric antigen receptor T-cell activity through a destabilizing domain switch and its chemical ligand

Background aimsDespite the impressive efficacy of chimeric antigen receptor (CAR) T-cell therapy, adverse effects, including cytokine release syndrome and neurotoxicity, impede its therapeutic application, thus making the modulation of CAR T-cell activity a priority. The destabilizing domain mutated from Escherichia coli dihydrofolate reductase (DHFR) is inherently unstable and degraded by proteasomes unless it is stabilized by its chemical ligand trimethoprim (TMP), a Food and Drug Administration-approved drug. Here the authors reveal a strategy to modulate CAR T-cell activity at the protein level by employing DHFR and TMP as a chemical switch system.MethodsFirst, the system was demonstrated to work in human primary T cells. To introduce the system to CAR T cells, DHFR was genetically fused to the carboxyl terminal of a third-generation CAR molecule targeting CD19 (CD19-CAR), constructing the CD19-CAR-DHFR fusion.ResultsThe CD19-CAR-DHFR molecule level was shown to be modulated by TMP. Importantly, the incorporation of DHFR had no impact on the recognition specificity and normal function of the CAR molecule. Little adverse effect on cell proliferation and apoptosis was detected. It was proved that TMP could regulate cytokine secretion and the in vitro cytotoxicity of CD19-CAR-DHFR T cells. Furthermore, the in vivo anti-tumor efficacy was demonstrated to be controllable through the manipulation of TMP administration. The approach to control CD19-CAR also succeeded in 19-BBZ(71), another CD19-targeting CAR with a different structure.ConclusionsThe proposed approach based on DHFR and TMP provides a facile strategy to bring CAR T-cell therapy under conditional user control, and the strategy may have the potential to be transplantable.