ADP-ribosylation reactions in Sulfolobus solfataricus,a thermoacidophilic archaeon

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87°C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of ¹⁴C incorporation from [¹⁴C]NAD on incubation at 80°C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine- ¹⁴C(U)]NAD and [adenine- ¹⁴C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40–100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

Detection of NAD: arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor

¹²⁵I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane (N-guanyltyramine), previously used to assay for the bacterial toxin choleragen (Mekalanos, J.J., Collier, R.J. And Romig, W.R. (1979) J. Biol. Chem. 254, 5894-5854) was utilized to identify NAD: arginine ADPribosyltransferases in animal tissues. The use of this radiolabelled ADPribose acceptor, rather than radiolabelled NAD, would bypass the problem posed by the almost ubiquitous presence of enzymes that degrade NAD. With a homogeneous ADPribosyltransferase from turkey erythrocytes, NAD and ¹²⁵I-labelled guanyltyramine as ADPribose acceptor, formation of ADPribosyl ¹²⁵-I-guanyltyramine was linear with time and enzyme concentration. The product was distinguishable on both thin-layer and high-performance liquid chromatography from that formed by cholerangen. Using ¹²⁵I-guanyltyramine, ADPribosyltransferase acitivity was also demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractioned, apparent activity was detected primarily in the microsomes. The NAD-dependent product of the microsomal reaction was, however, distinguished from the turkey erythrocyte transferase by thin-layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free ¹²⁵I on high-performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP and NADPH. Phenyl boronate selectively bound ADPribosyl ¹²⁵I-guanyltyramine and other metabolites of ¹²⁵I-guanyltyramine which were formed by microsomes in a NAD-dependent process. These metabolites were distinguished from ADPribosyl ¹²⁵I-guanyltyramine by high-performance liquid chromatography. These results indicate that in some cases, for example, turkey erythrocyte cytosolic and membrane fractions, ¹²⁵I-guanyltyramine can be used to quantify ADPribosyltransferases in crude mixtures, whereas in others, for example, rat liver microsomes, high-performance liquid chromatographic analysis must be used to identify products.     

ADPRibosylation of chicken red cell tubulin and inhibition of microtubule self-assembly in vitro by the NAD(+)-dependent avian ADPRibosyl transferase.

Chicken erythrocyte tubulin was found to undergo NAD(+)-dependent ADPribosylation in vitro in the presence of ADPRtransferase also isolated from avian red blood cells. Unlike the low level of ADPR incorporation catalyzed by Cholera and Pertussis toxins (i.e., less than 0.005 mol ADPR/mol tubulin), the avian system displayed a much higher stoichiometry of 0.8-1.2 mol ADPR/mol tubulin. Modification resulted in potent inhibition of microtubule self-assembly, even in the presence of bovine brain microtubule-associated proteins or with the addition of pre-assembled microtubules.     

Characterization of specific differences in protein phosphorylation of the plasma membrane and the endoplasmic reticulum of mouse fibroblasts

Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [γ-³²P]ATP and [γ-³²P]GTP as precursors. With ATP maximum overall incorporation of ³²P into both membrane fractions occured at pH 7.8 in the presence of 10 mM MgCl₂ after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of ³²P at pH 7.8 and 10 mM MgCl₂ after incubation for 3 min.The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phophorlation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular wieght of about 100 000 and the ATP-specific main component a molecular weight of 110 000.The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl₂ concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (M_r = 110 000) was no more phosphorylated whereas with GTP the main component M_r = 100 000 was essentially the sole phosphorylated protein.     

Poly(ADPRibose) levels as a function of chick limb mesenchymal cell development as studied in vitro and in vivo.

NAD is converted into a chromatin-bound polymer, poly(ADPribose), with the excision of nicotinamide. In intact cells, the incorporation of labeled adenine, through NAD, into poly(ADPribose) has been correlated with the commitment and/or initial phenotypic expression of chick limb mesenchymal cells. Using an assay for chemical quantities of poly(ADPribose), we report here measurements of poly(ADPribose) during limb development in situ and during limb mesenchymal cell commitment and expressional events in cell culture. Substantial changes in the levels of poly(ADPribose) are observed during early phases of limb cell development either in situ (embryonic stages 22 to 26) or in culture (Days 1 to 4); during this time, we observed a threefold decrease in poly(ADPribose) per unit DNA (21 to 7 nmoles/mg DNA), as compared to relatively minor changes of 10 to 20% during later expressional events especially related to muscle development. These observations establish a correlation between cellular poly(ADPribose) levels and the early phases of chick limb mesenchymal cell differentiation and development.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca and calmodulin

The effects of Ca²⁺ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [³²P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca²⁺. Maximal incorporation of [³²P] into the protein was achieved with free Ca²⁺ concentrations of 90 μM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 μM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [³²P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca²⁺ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca²⁺ concentrations between 10⁻⁶ – 10⁻⁵ M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca²⁺ and calmodulin.     

Light or high (30°C) temperature effects on l-[U-C]leucine incorporation and protein patterns in embryos and endosperm during the early phase of germination of the negatively photoblastic and thermosensitive seeds of Phacelia tanacetifolia

The activities of l-[U-¹⁴C]leucine uptake and incorporation into proteins of embryos and endosperm of seeds of Phacelia tanacetifolia Benth. cv Bleu-Clair were analysed during the first 24 h of incubation under conditions optimal for germination (16°C in darkness) and in two inhibitory conditions: 16°C in the light and 30°C in darkness. Blocking germination induced by light or 30°C was accompanied by the inhibition of l-[U-¹⁴C]leucine uptake and incorporation in embryos. In the endosperm, the activation of l-[U-¹⁴C]leucine uptake was of the same magnitude for the non-inhibited and the light-inhibited seeds and much higher for the 30°C-inhibited seeds; the activation of l-[U-¹⁴C]leucine incorporation was quantitatively similar in all three conditions, with the patterns of newly synthesised proteins qualitatively different in the endosperm from light- or 30°C-inhibited seeds. The results showed that germination of P. tanacetifolia seeds is controlled by light or super-optimal temperature through the inhibition of the activation of transport and protein synthetic activities in embryo without effect on the endosperm. We suggest, on the basis of the translational activity, the possibility that in the inhibitory conditions the blockade of the embryo to operate as a sink affects the transition of the endosperm to operation as a source.     

ADP-ribosylation of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6

Poly(ADP-ribosylation) [poly(ADPR)] is a posttranslational modification of chromosomal proteins that affects the structural and functional properties of chromatin. We have studied poly(ADPR) of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6 (clone 41) by a combination of affinity chromatography on phenylboronate and immunoblotting with monoclonal antibodies against poly(ADPR) chains and polyclonal antibodies against ADPR-transferase and topoisomerase I, respectively. Constitutive, steady-state poly(ADPR) substitution of ADPR-transferase was estimated at 4% and that of topoisomerase I at 0.1%. Active oxygen produced extracellularly by xanthine-xanthine oxidase and the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine transiently increased the level of poly(ADPR) substitution of these enzymes by a factor of 6-10. While the poly(ADPR) substitution of ADPR-transferase remained elevated after 60 min of incubation, the poly(ADPR) substitution of topoisomerase I had returned to control values within this time. Benzamide (100 microM) partially prevented the stimulation of poly(ADPR) synthesis by these agents. We speculate that self-inactivation of ADPR-transferase by poly(ADPR) represents a feedback mechanism that has the function to avoid excessive poly(ADPR) synthesis and concomitant NAD and ATP depletion. Inactivation of topoisomerase I in the neighborhood of DNA breakage may temporarily shut down DNA replication and allow DNA repair to occur.     

Effect of 2,4-D on Cell Wall Metabolism of Peroxyacetyl Nitrate Pretreated Avena Coleoptile Sections

Avena coleoptile sections were exposed to nonlethal concentrations of peroxyacetyl nitrate (PAN). The sections were then incubated in solutions of 50 mM glucose plus 2.5 mM poassium phosphate with various concentrations of 2,4-dichlorophenoxycetic acid (2,4-D). Growth after 4 hours was measured. A corresponding series of experiments was carried out with glucose-¹⁴C (U) in the subsequent incubation medium and the effect of the 2,4-D treatments on ¹⁴C incorporation into various cell wall components was determined. Growth in the PAN-treated sections, although still partially inhibited, was greater at auxin levels normally superoptimal for growth than at the former optimum. Incorporation into all cell wall fractions was similar to growth in the case of control treated tissue. Most of the cell wall constituents, but particularly cellulose and less soluble noncellulosic polysaccharides, tended to show higher incorporation at the levels where PAN-treated growth was also higher. It was concluded that effects by PAN on cell wall metabolism in growing tissue are similar to the effects on growth and that the mechanism of alleviation of growth inhibition is probably through decreased inhibition of wall metabolism.     

Diphtheria toxin inhibits the synthesis of myelin proteolipid and basic proteins by peripheral nerve in vitro

Abstract— Diphtheria toxin (DT) did not produce measurable degradation of myelin proteins or sulphatide in sciatic nerves of chick embryos after incubation in vitro for 4 h. In contrast, DT inhibited the in vitro incorporation of L-[U-¹⁴C]leucine into myelin proteins by the nerves after a delay of 1 h. Separation of the myelin proteins by SDS-polyacrylamide gel electrophoresis indicated that the synthesis of Wolfgram proteins and proteins not entering the gel was inhibited by 21–22 per cent, whereas synthesis of myelin proteolipid and basic proteins was inhibited by 79–88 per cent. Incorporation of ³⁵SO₄ into myelin [³⁵S]sulphatide was also inhibited by DT after a delay of 2 h. The inhibition of [³⁵S]sulpha-tide incorporation into myelin caused by DT differed from that observed with puromycin in that it did not depend on depletion of an intracellular transport lipoprotein. Instead, the inhibition seemed to be secondary to the decreased synthesis of myelin proteolipid and basic proteins.     

Effect of “osmotic stress” on protein and nucleic acid synthesis in isolated tobacco protoplasts

The incorporation of labeled precursors into RNAs and proteins of isolated tobacco (Nicotiana tabacum L.) leaf protoplasts decreases with increasing osmotic pressure in the incubation medium. The incorporation of precursors into RNA and proteins is linear for 15–18 h after the isolation of the protoplasts, irrespective of the osmolarity of the culture media. The uptake of precursors is also affected by the osmolarity of the medium. However, the osmotic stress-induced inhibition of incorporation of precursors into RNA and proteins is also apparent if the differences in uptake are taken into consideration in the calculation. Incorporation of ³²P into TMV-RNA is also inhibited by osmotic stress. As assayed by the double labeling ratio technique, osmotic stress has less unequivocal effect on TMV protein synthesis.Abbreviations PP protoplast - RNase ribonuclease - rRNA ribosomal ribonucleic acid - SDS sodium dodecyl sulfate - SSC 0.1 M Na-acetate in 0.15 M NaCl - TCA trichloroacetic acid - TMV tobacco mosaic virus     

Metabolic fate of extracellular NAD in human skin fibroblasts

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.     

Metabolism of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes from rat brain

Abstract— Synthesis of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes isolated from rat brain was investigated and compared with the synthesis of these compounds in microsomes and mitochondria. Electron microscopic and marker enzyme studies showed the contaminants in the synaptosomal preparation to consist of a few microsomes and almost no free mitochondria. In synaptosomes, addition of 1,2-diglyceride exerted no effect on the incorporation of [¹⁴C]choline into phosphatidylcholine or on the incorporation of [³H]myo-inositol into phosphatidylinositol, but it stimulated the incorporation of CDP[1,2-¹⁴C]choline into phosphatidylcholine by more than 50 per cent. The incorporation of the latter in intact synaptosomes, lysed synaptosomes and purified mitochondria was 15-6, 27 and 9-9 per cent, respectively, of that in the microsomes. The incorporation of [³H]myo-inositol into the phosphatidylinositol of synaptosomes and purified mitochondria was 15-8 and 11-1 per cent, respectively, of that in the microsomes. Maximal incorporation of [³H]myo-inositol occurred at pH 7–5 in a medium containing Mg²⁺ and CTP; it was linear with time and protein concentration and was inhibited by 1 mM Ca^{2 +} but unaffected by the presence of ATP. This incorporation of myo-inositol appeared to occur through the reversal of the CDP-diglyceride: inositol transferase reaction. The demonstration of carnitine palmityl transferase in synaptosomes indicated that, as in mitochondrial and erythrocyte membranes, fatty acids can be transported across the synaptosomal membrane. In contrast to mitochondria where maximal incorporation of [¹⁴C]carnitine into palmityl carnitine was observed after 20 min of incubation, the incorporation in synaptosomes increased as a function of time up to 60 min of incubation. We conclude that synaptosomes can carry on de novo synthesis of lipids, although at a limited rate. From the present data we cannot state with certainty how much of this synthesis is attributable to membranes originating from the endoplasmic reticulum.     

NAD turnover during early development of Xenopus laevis

The NAD pools of Xenopus laevis oocytes and early embryos can be radioactively labelled by microinjection of [adenine- ³H]NAD. This technique is used to study the metabolism of NAD in oocytes and during early development. The rate at which NAD is degraded in vivo has been monitored by determining the rate of transfer of adenine residues from the NAD pool into other nucleotides and polynucleotides. In oocytes, NAD turnover is extremely slow, with a half-life of about 400 h. NAD turnover increases dramatically after fertilisation, and the half-life of the compound decreases to 37 h in 5-h-old embryos and to 10 h in 40-h-old embryos. 2 mM 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, reduces the NAD turnover rate by about 20%, whereas 5 mM isonicotinic acid hydrazide, a specific inhibitor of NAD glycohydrolase, produces no significant inhibition. This indicates that a significant fraction of the considerable NAD turnover observed involves poly(ADP-ribose) polymerase. Our results indicate that poly(ADP-ribose) polymerase is active during early development and suggest that this activity may be involved in one or more aspects of the nuclear metabolism of the embryo.     

EVIDENCE FOR INVOLVEMENT OF Ca2+ IN THE INCORPORATION OF 32Pi INTO THE PHOSPHATIDYLINOSITOL OF RAT DIAPHRAGM

Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of ³²P_i into phosphatidylinositol (PI) in rat diaphragm incubated in Ca²⁺-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of ³²P_i into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Ca^a+-chelating properties; the inhibition of the incorporation of ³²P_i could be completely reversed by the addition of excess Ca²⁺ but not Mg²⁺. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca²⁺, rather than extracellular Ca²⁺, was involved in the effect. In contrast to its inhibition of the incorporation of ³²P_i EGTA enhanced the incorporation of [³H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [³Hlinositol by the tissue. Our results suggest that the level of intracellular Ca²⁺ plays a role in the regulation of the incorporation of ³²P_i into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of ³²P_i into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of ³²P_i into α-glycerophosphate.     

NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties

NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent K _m for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn²⁺ and Ca²⁺ could be substituted for Mg²⁺. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a K _i of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.This paper is dedicated to Sydney C. Rittenberg on the occassion of his retirement, with respect and much affection, in appreciation for his friendship and years of distinguished service as a teacher and scientist     

Effect of heparin modification on its circular dichroism spectrum

The sarcoplasmic reticulum and glycogen pellet derived from rabbit skeletal muscle and the sarcolemma and sarcoplasmic reticulum from pig skeletal muscle contains NAD:dependent mono ADP-ribosyltransferase activity toward the guanidine analog, P- nitrobenzylidine aminoguanidine. No or little activity could be found in the sarcolemma or sarcoplasmic reticulum derived from canine cardiac muscle. Seventy percent of activity extracted from rabbit skeletal muscle is localized in the sarcoplasmic reticulum. The enzyme has a pH optimum of 7.4, and KM of 0.5 mM and 0.35 mM for NAD and p-nitro benzylidine aminoguanidine, respectively. Inorganic phosphate, KCl, and guanidine derivatives inhibit the reaction. Incubation of the sarcoplasmic reticulum or glycogen pellet with (adenylate-32P) NAD or [adenosine-14C(U)]-labeled NAD results in the incorporation of radioactivity into proteins. A large number of proteins are labeled in the sarcoplasmic reticulum fraction. The major labeled band in the glycogen pellet corresponds to a protein of molecular weight of 83 K.