Guanido Group Research Articles

CD studies of synthetic polypeptides as histone models: Poly(L‐Arg‐L‐Ile‐Gly), poly(L‐Arg‐L‐Nva‐Gly), and poly(L‐Arg‐L‐Nle‐Gly)

AbstractSequential polypeptides (L‐Arg‐X‐Gly)n were prepared as synthetic models of arginine‐rich histones to study their structure and their stereospecific interactions with DNA. In our previous work the conformational characteristics of poly(L‐Arg‐L‐Ala‐Gly), poly(L‐Arg‐L‐Val‐Gly), and poly(L‐Arg‐L‐Leu‐Gly) have already been analyzed. To obtain further insight into the influence of the X residue side chain on the conformation of the (L‐Arg‐X‐Gly)n polytripeptides, we now report their synthesis and cd properties when X represents the amino acid residues Ile, Nva, and Nle. The pentachlorophenyl active esters of the appropriate tripeptides were used to perform the polymerization, and the toluene‐4‐sulfonyl group was used to protect the arginine guanido group. CD spectroscopy showed that, in 100% trifluoroethanol, the degree of helical conformation increased in the order Ile → Nle → Nva. An equilibrium between β‐turn, α‐helix, and random‐coil conformers occurred in 100% hexafluoroisopropyl alcohol, while a rise in the temperature or the addition of water favored the α‐helix, the highest percentage of which was observed in a mixture of hexafluoroisopropyl alcohol: water (20 : 80) and in the order Ile → Nle → Nva. In aqueous solutions (at pH 7 and 12) the polymers behaved as a random coil, but they were forced to a less aperiodic structure, over a range of ionic strengths (0–0.5M NaF). A rise in temperature of up to 70°C in 100% trifluoroethanol resulted in a decrease of the α‐helix percentage of the polymers, while in aqueous solutions the aperiodic structure decreased with increasing temperature. This study proved the importance of the nature of the X residue (length, Cβ branching) in relation to the structural order of the sequential polypeptides. We concluded that the polymers prepared are suitable models for arginine‐rich histones.

Biosynthesis of pyrimidine nucleotides and arginine in a suspension culture of Catharanthus roseus

1. 1. The intracellular levels of pyrimidine nucleotides and of arginine changed markedly during the growth cycle (10 days) of Catharanthus roseus in suspension culture. 2. 2. The levels of UTP and UDP-glucose increased rapidly during the 24 hr which followed transfer of the cells to fresh Murashige-Skoog medium. The levels of other pyrimidine nucleotides also increased after the transfer of cells, but the maximum level of UMP was detected on day 5. 3. 3. The level of free arginine decreased just after subculture, although the level of arginine, which had been incorporated into protein, increased during the logarithmic phase of cell growth. Ornithine could not be detected at any stage of growth of the cells. 4. 4. The label from NaH 14CO 3 was incoprorated into pyrimidine nucleotides and into the guanido group of arginine. Most of the radioactivity of these compounds was associated with nucleic acids and proteins. 5. 5. The contribution of de novo synthesis of pyrimidine nucleotides was higher in the early phases of cell growth than in the stationary phase. In contrast, the maximum incorporation of NaH 14CO 3 into arginine was found during the cell division phase when active synthesis of proteins occurred. 6. 6. The mechanisms which regulate the biosynthesis of pyrimidine nucleotides and arginine in vivo are discussed.

Specific targets of alkylating agents in nuclear proteins of cultured hepatocytes

We have established a specific correlation between the carcinogenic potency of a series of alkylating agents, with a mechanism of reaction ranging between Ingold's S N1−S N2 (ENU MNU = MNNG EMS DMS = MMS) (Vogel et al., 1979; Bartsch et al., 1983) and specific target sites in the amino acids of nuclear proteins of cultured hepatocytes. More potent carcinogens, that react predominantly with an Ingold's S N1 mechanism, mainly alkylate the amino group of lysine and the guanido group of arginine. Weaker carcinogens, reacting with a mechanism closely resembling an Ingold's S N2, mainly alkylate the sulfhydryl group of the cysteine and the 3 position of the imidazolic ring of histidine. A compound with an intermediate type of reactivity alkylates, to a comparable extent, all 4 of the above-described positions. Although stable DNA damage brought about by alkylating carcinogens is considered to be the most likely cause of neoplastic transformation, epigenetic modifications may also play an important role in the process, especially because of their extreme stability. We have verified the existence of a linear correlation between the Swain-Scott substrate constant ( S) of each compound and the amount of alkylation produced at the specific target sites. This type of correlation could be the basis of a ‘short-term’ genotoxicity assay in a battery of complementary tests.

Macrophage Cytotoxicity: Role for L-Arginine Deiminase and Imino Nitrogen Oxidation to Nitrite

Previous studies have shown that cytotoxic activated macrophages cause inhibition of DNA synthesis, of mitochondrial respiration, and of aconitase activity in tumor target cells. An L-arginine-dependent biochemical pathway synthesizing L-citrulline and nitrite, coupled to an effector mechanism, is now shown to cause this pattern of metabolic inhibition. Murine cytotoxic activated macrophages synthesize L-citrulline and nitrite in the presence of L-arginine but not D-arginine. L-Citrulline and nitrite biosynthesis by cytotoxic activated macrophages is inhibited by NG-monomethyl-L-arginine, which also inhibits this cytotoxic effector mechanism. This activated macrophage cytotoxic effector system is associated with L-arginine deiminase activity, and the imino nitrogen removed from the guanido group of L-arginine by the deiminase reaction subsequently undergoes oxidation to nitrite. L-Homoarginine, an alternative substrate for this deiminase, is converted to L-homocitrulline with concurrent nitrite synthesis and similar biologic effects.

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies.

AbstractThe sequential polypeptides (L‐Arg‐X‐Gly)n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine‐rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene‐4‐sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L‐Arg‐X‐Gly)n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β‐structure. Characteristics of β‐turn are excluded from the poly(L‐Arg‐L‐Leu‐Gly), which assumed the most pronounced helical conformation. The poly(L‐Arg‐L‐Val‐Gly) exerts a significant preference to the β‐turn structure compared to that of poly(L‐Arg‐L‐Ala‐Gly). Thus the probability for helical, β‐structure or β‐turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X‐residue side chain (β‐branching). We concluded that the prepared sequential arginine‐containing polypeptides are plausible models for histone fractions, f3 and f2α1.

Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethyl-cellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit onA. oryzae,B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive

Affinity labeling of dihydrofolate reductase with an antifolate glyoxal.

Dihydrofolate reductases from different species contain several highly conserved arginines, some of which have been shown by x-ray crystallography to have their guanido groups near the p-aminobenzoyl glutamate moiety of enzyme-bound methotrexate. The orientation of one of these (Arg-52) appears to be completely reversed in comparing the crystal structures of Escherichia coli with Lactobacillus casei enzyme (Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., and Kraut, J. (1982). J. Biol. Chem. 257, 13650-13662). We synthesized a novel antifolate containing a glyoxal group designed to react specifically with active-site guanido groups which are able to approach the p-aminobenzoyl carbonyl of methotrexate. The binding of this compound to the enzyme was competitive with dihydrofolate (DHF) in ordinary buffers. In borate buffer at pH 8.0 it inactivated dihydrofolate reductases from both E. coli and L. casei at similar maximum rates, while the chicken liver enzyme was more slowly inactivated. The inactivation was stoichiometric, paralleled the loss of the glyoxal chromophore, and showed saturation kinetics. Inhibitor binding and thus inactivation was enhanced by NADPH, while DHF protected the enzyme. This allowed calculation of the Kd for DHF which was found to be identical with its Km. The stoichiometrically inactivated enzyme displayed the 340-nm chromophore characteristic of 4-aminopteridines bound to dihydrofolate reductase confirming active-site labeling with normal orientation of the ligand. The ligand remained covalently bound to inactivated enzyme upon denaturation at low pH but dissociated at neutral pH. Computer graphic modeling of the crystal structures predicted reaction of Arg-31 but not Arg-52 in L. casei dihydrofolate reductase and of only Arg-52 in the E. coli enzyme. Purification of the CNBr fragments from the inactivated enzymes gave a single labeled peptide for each species. The particular peptide tagged in each case was unaffected by the presence of NADPH and was in excellent agreement with the crystallographic predictions.

Arginine-rich proteins in spherical inclusions of human locus coeruleus neurons demonstrated by benzil modification.

Previous studies have shown that aminergic neurons in the normal human brain contain acidophilic cytoplasmic inclusions--called protein bodies (PBs)--that are reduced or absent in parkinsonism and disrupted in depression. The purpose of the present study was to elucidate the constitution of PBs in five formalin-fixed normal human brains using histochemical methods specific for histones, protamines, and the amino acid arginine. PBs were revealed with alkaline fast green and bromphenol blue, exhibiting a high content in histones and in protamines. They developed blue metachromasia with phosphotungstic acid-hematoxylin and green fluorescence with phenanthrenequinone, which established the presence of arginyl residues. Using benzil, which selectively modifies the guanido group of arginine, staining was blocked for each of the above two methods. The application of Mallory's trichrome procedure after benzil differentiated the PBs into an unstained core and a still fuchsinophilic rim. Since the fuchsinophilia of the rim was shown to persist after acetylation as well, we suggest that this rim probably contains acidic macromolecules that attach to the basic charges of the amphoteric acid fuchsin. We conclude that the PB are complex structures consisting of a core segregating arginine-rich proteins and a rim which probably contains macromolecules of an acidic nature.

Autophosphorylation of type 2 casein kinase TS at both its α- and β-subunits: Influence of different effectors

Rat liver casein kinase TS (Ck-TS) having quarternary structure α 2β 2, autophosphorylates at its 25 kDa, β-subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck-TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β-subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β-subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β-subunit, while promoting the autophosphorylation of the 38 kDa α-subunit. In the presence of such polyarginyl effectors the 130 kDa Ck-TS is converted into forms with higher sedimentation coefficient.

Role of charged groups in factor XI/XIa activity.

To elucidate the role of charged groups in expression of factor XI coagulant activity, the charged groups of purified human blood coagulation factor XI/XIa containing 125I-XI/XIa were derivatized: free amino groups by succinylation, guanido groups of arginine by reaction with phenylglyoxal hydrate, and free carboxyl groups by reaction with ethylenediamine. The modified proteins were tested for: 1) ability to adsorb to glass, 2) ability to be cleaved by trypsin or factor XII-high molecular weight kininogen, 3) coagulant activity. The amino group-modified factor XI had a significantly decreased ability to bind to glass; modification of arginine or carboxyl groups did not affect adsorption. Trypsin cleaved factor XI with modified free amino, guanido, or carboxyl groups. Factor XII-high molecular weight kininogen could cleave only the arginine-modified factor XI. Amino group-modified factor XI and carboxyl group-modified factor XI lost all their factor XI assay activity, whereas arginine-modified factor XI retained 50% of the original activity. Amino group-modified factor XI could not be activated by trypsin, but arginine-modified and carboxyl group-modified factor XI could be activated by trypsin to 50% of the original activity. Succinylation of the amino groups of factor XIa destroyed all its factor XIa activity. Arginine-modified and carboxyl group-modified factor XIa retained 50% of their factor XIa activity. We conclude that epsilon-amino groups are essential for adsorption; activation by factor XII-high molecular weight kininogen requires free amino and carboxyl but not guanido groups; free amino, carboxyl, and guanido groups in factor XIa all appear to be critical for interaction of factor XIa with factor IX.

Synthetic fragments of protamines as model substrates for rat liver cyclic AMP-dependent protein kinase

Nine synthetic peptides reproducing either exactly or with suitable substitutions three phosphorylatable sites of the protamines thynnine Z1 and galline have been prepared and tested as phosphate acceptors for the rat liver cyclic AMP-dependent protein kinase (type I). The most significant results obtained can be summarized as follows: 1. The hexapeptide: Arg-Arg-Ser-Thr-Val-Ala gives two phpsphorylated products, containing only Ser-P and both Ser-P and Thr-P, respectively. The relative amount of the di-phosphorylated derivative is not dependent on the incubation time but rather on the concentration of the substrates. 2. Both the replacements of ornithine for the two N-terminal arginines of glutamic acid for Val 5 in the above peptide completely prevent phosphorylation, without conferring any inhibitory activity to the modified peptides, thus supporting the view that the N-terminal guanido groups and the C-terminal hydrophobic residue(s) are both required for the binding at the catalytic site rather than for the subsequent transphosphorylation reaction. 3. The replacement of alanine for Ser 3 gives rise to a peptide whose Thr 4 residue is still phosphorylated with an efficiency comparable to that of the unmodified peptide. The di-substituted peptide: Arg-Arg-Ala-Ser-Val-Ala however exhibits a dramatically lower K m value indicating that serine is a much better target than threonine whenever it is not adjacent to the N-terminal arginine couple. 4. The importance of the distance between the target residue and the N-terminal basic determinants is also evidenced by the phosphorylation of the dodecapeptide: Pro-(Arg) 5-Ser-Ser-Arg-Pro-Val-Arg exhibiting a K m value about 20-times higher than that of salmine A1, whose phosphorylation site is comprised within an identical amino acid sequence, including however three rather than two adjacent serine residues. 5. The tetradecapeptide: (Arg) 4-Tyr-Gly-Ser-(Arg) 6-Tyr is completely unaffected by the kinase though a very similar site is found phosphorylated in native iridines, probably through a cyclic AMP-independent mechanism.

Agglutination of human erythrocytes by Fusobacterium nucleatum: factors influencing hemagglutination and some characteristics of the agglutinin.

Various aspects of agglutination of human erythrocytes by fusobacterium nucleatum were examined. Titration experiments done in buffered saline at pH 6 to 10 showed the same agglutination endpoint. The presence of high concentrations of NaCl in reaction mixtures did not alter titers, but KCl in concentrations of 0.5 to 3.6 M increased titers twofold. The agglutinin was inactivated by heat, acid, alkali, 5% Formalin, and the proteolytic enzyme subtilopeptidase A and therefore appeared to be a protein. Treatment of bacterial cells with 2-mercaptoethanol had no effect. Hemagglutination inhibition experiments revealed that arginine and other compounds containing a guanido group as part of their structure were inhibitory at low concentrations. Various hexoses, some hexose derivatives, and most common metal ions, when added to bacterium-erythrocyte mixtures, had no effect. The binding of dansyl-L-arginine to bacteria, but not erythrocytes, was demonstrable by ultraviolet fluorescence microscopy.

Mouse Macrophage Arginase

SummaryMouse peritoneal macrophages in culture released a macromolecular factor capable of inhibiting mouse L1210 lymphoma cells in vitro. The factor was proved to be arginase by several criteria. The inhibition was reversed by adding arginine or citrulline to culture, the arginine in the culture medium was consumed, and the guanido group of arginine was hydrolyzed exclusively to urea as demonstrated by electrophoresis and chromatography. The factor has a molecular weight of 110,000 and a Km value of 1.0 × 10-2M. These and other parameters, such as a requirement for Mn2+, alkaline pH optimum, substrate specificities, and inhibition by ornithine, were similar to those of liver arginases. The arginine in the medium was decomposed by first-order kinetics, and the arginase activity in the supernatant of 10 × 106 peritoneal exudate cells/ml was enough to deplete arginine in the medium to below the level required for L1210 cells to grow in culture.Arginase activity was also detected in the peritoneal washing fl...

Neuroactive properties of some phosphonic analogues of natural guanido compounds

1. 1. 2-Guanidoethylphosphonic acid and guanidomethylphosphonic acid, the phosphonic analogues of taurocyamine and glycocyamine, were applied by microiontophoresis to rat cerebral and cerebellar neurones. 2. 2. 2-Guanidoethylphosphonic acid has an inhibitory effect similar to that produced by β-guanidopropionic acid, taurocyamine and also the parent compound, β-alanine. 3. 3. In contrast to the inhibitory effect of glycocyamine, the excitatory action of guanidomethylphosphonic acid (which was also observed with S-(phosphonomethyl)-thiourea) may be related to a particular conformation of these molecules produced by the interaction between the strongly acidic phosphonic group and the guanido group.

Ambiguities in the enzymology of sulfur-containing compounds

In the course of this meeting we have occasionally met reactions involving a variety of sulfur-containing compounds carried out by systems or enzymes also used for different purposes. In this final lecture we should like to point out how this phenomenon is much more general than believed, thus indicating the occurrence of a real ambiguity in the enzymology of sulfur-containing compounds. This fact became to the attention of one of us early in 1956, when cystamine (I) and lanthionamine (II) were assayed as substrates for diamine oxidase (1). It was known at that time that diamine oxidase was an enzyme of broad specificity, being able to use as substrates diamines having different carbon chain lengths and also diamines with one of the amino groups substituted with an imidazole or a guanido group (2). However it was not known whether a substantial change of the chemical composition would have impaired the enzymatic attack on the substrate. It was therefore a surprise to find an enzyme unable to recognize the substitution of a methylene carbon of the substrate with one or two atoms of sulfur. Diamine oxidase uses not only I and II as substrates but also lanthionamine sulfone, although at a lower rate than I and II (1). The interest of this finding was increased by the observation that the rate of the oxidation of I and II was in the range of that of the traditional substrates and that the oxidation was followed by the cleavage of the intermediate cystaldimine (IV) leading to the formation of compounds of biological interest like thiocysteamine (VI), hypotau- rine (VII), thiotaurine (VIII) and taurine (IX).

Involvement of arginine residues in the binding site of cholera toxin subunit B

Modification of one or two of three guanido groups in the binding subunit (B) of cholera toxin was achieved at pH 9 with cyclohexanedione at 50 mM or 150 mM concentration, respectively. No change in the helix content or the pentameric structure was observed in the process. The ability to form precipitate with ganglioside G ml or anti-cholera toxin antibody was abolished only when two of the three arginine residues were modified. Analyses of Arg-containing peptides revealed that reaction with cyclohexanedione occurred first with Arg-73 and then with Arg-35. This suggests that Arg-35 or the region in proximity is involved in the interaction of subunit B with ganglioside G ml or the antibody. A diagram of secondary structure as predicted by the Chou-Fasman rule indicates that this region is within a long stretch of β-sheet configuration.

The presence of citrulline in epidermal proteins

Citrulline is present in the stratum corneum proteins of human, cow snout, pig snout and guinea pig epidermis but is absent from the stratum corneum proteins of frog, mouse, turtle, rat and hamster epidermis. The amino acid is released by acid hydrolysis and ranges from 1.7 to 5.5 residues per thousand residues of protein amino acid. Protein derived citrulline co-chromatographs with authentic l-citrulline on an amino acid analyzer, on Dowex-50, on Dowex-2 and on thin-layer chromatography. Dansylated material co-chromatographed with authentic dansyl- l-citrulline in two thin-layer chromatography systems. Labelling experiments have shown that the protein bound citrulline is derived from protein bound arginine and probably results from enzymatic conversion of the guanido group to the ureido group.

Interaction of plasma lipoproteins containing apolipoproteins B and E with heparin and cell surface receptors

Low density lipoproteins (LDL) containing apolipoprotein B and a high density lipoprotein (the HDL c) containing apolipoprotein E bind to the same cell surface receptors of fibroblasts and also bind to and are precipitated by heparin. To determine if the nature of the chemical interaction of these lipoproteins with the receptor and with heparin were identical, we undertook to compare the effects of the chemical modification of selected amino acid residues of these lipoproteins on receptor and heparin binding activities. Previously, we reported the importance of the positively charged amino acids arginine and lysine in the interaction of LDL and HDL c with the cell surface receptors. Lysine residues were modified by a procedure which did not alter the positive charge on the ϵ-amino group (reductive methylation) or by procedures which neutralized this positive charge (acetoacetylation and carbamylation). Modification of arginyl residues was accomplished using 1,2-cyclohexanedione. Whereas all procedures used to modify the arginine and lysine residues totally abolished receptor binding activity, only the procedures which neutralized the positive charge of these residues abolished heparin binding. Reductive methylation of the lysine residues, which preserved the positive charge on the ϵ-amino group, did not significantly prevent heparin binding or heparin precipitation of LDL or HDL c. Several conclusions are possible from the results of these studies. The protein moieties of both the LDL (apolipoprotein B) and HDL c (apolipoprotein E) can interact with heparin. Furthermore, both arginine and lysine residues participate in the interaction of these lipoproteins with heparin,apparently through an ionic interaction between the positively charged guanido group of arginine or the ϵ-amino group of lysine and the heparin. Although the same residues (arginine and lysine) are involved in cell surface receptor binding of these lipoproteins, the nature of the chemical interaction appears to be different and does not appear to be strictly ionic.

Topochemistry of the binding of phosphotungstic acid to collagen

The interaction between collagen and phosphotungstic acid can be divided into two specific reaction steps, dependent on the steric arrangement. 1. (1) A relatively quick, firm intersubfibrillar binding (within ≈ 10 min) of the polyanions PW 12O 40 3− to easily accessible basic groups. This occurs even when the units become more compact through tension, as indicated by meridional layer-line reflections. 2. (2) A less tight, intrasubfibrillar bond to relaxed incubated fibres (≧ 20 min) with participation of relatively heavy accessible amino groups, indicated by an equatorial reflection at ~19 Å. The intrasubfibrillar PW 12O 40 3− deposition is restricted by previous aldehyde cross-linking of the fibres, whereas it continues further with non-drawn and non-crosslinked fibres. In the latter case, a contraction of the fibre and a shortening of the 670 Å period are observed. The polyanion PW 11O 39 7− present at pH 5.5 to 7 is evidently bound to relaxed fibres by the guanido group only. The scattering reflections, the contraction of phosphotungstic acid-treated fibres and the associated changes of the long period, are interpreted on the basis of an eight-stranded unit for the subfibrils. These results have been obtained with native and chemically modified collagen fibres using conventional and synchrotron X-ray radiation as well as mechanical measuring devices.

Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase

Mitochondrial NADH dehydrogenase has been purified to homogeneity by resolution of Complex I from beef heart mitochondria with the chaotrope NaClO 4 and precipitation of the enzyme with ammonium sulfate. The enzyme is water-soluble, has a molecular weight of 69,000 ± 1000 as determined by gel filtration on Sephadex G-100 and agarose 1.5 M. It is an iron-sulfur flavoprotein, with the ratio of flavin (FMN) to nonheme iron to labile sulfide being 1:5–6:5–6. The FMN content suggests a minimum molecular weight of 74,000 ± 3000 for the enzyme. NADH dehydrogenase is composed of three subunits with apparent M r values, as determined by acrylamide gel electrophoresis as well as by gel filtration on agarose 5 M both in the presence of sodium dodecyl sulfate, of about 51,000, 24,000, and 9–10,000. Coomassie blue stain intensities of the subunits on acrylamide gels suggest that they are present in NADH dehydrogenase in equimolar amounts. However, summation of the apparent M r values of the dodecyl sulfate-treated subunits appears to overestimate the molecular weight of the native enzyme. The amino acid compositions of NADH dehydrogenase and of each of the isolated and purified subunits have been determined. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH by quinones, ferric compounds, and NAD (3-acetylpyridine adenine dinucleotide was used). All the activities of NADH dehydrogenase are greatly stimulated by addition of guanidine (up to 150 m m), alkylguanidines, arginine, and arginine methyl ester to the assay medium. Phosphoarginine had no effect. These results pointed to the importance of the positively charged guanido group, which appears to interact with and neutralize the negative charges on NAD(P)H and thereby allow for better enzyme-substrate interaction. In the absence of guanidine, NADPH is essentially unoxidized by the enzyme at pH values above 6.0. However, both NADPH dehydrogenase and NADPH → NAD transhydrogenase activities increase dramatically as the assay pH is lowered below pH = 6. Since the p K of the 2′-phosphate of NADPH is 6.1, it appears that the above pH effect is related to protonation of the 2′-phosphate, thus rendering NADPH a closer electronic analog of NADH, which is the primary substrate of the enzyme.