Amino Acid-modifying Reagents Research Articles

Extracellular lysine 38 plays a crucial role in pH-dependent transport via human monocarboxylate transporter 1

Human monocarboxylate transporters (hMCTs) are expressed in many tissues and mediate the transport of various substrates across the plasma membrane. Among hMCTs, hMCT1–4 cotransport H+ with monocarboxylates such as pyruvate and l-lactate, implying that these proteins recognize both substrate and H+. However, the mechanism of translocation, and particularly that of hMCT1 pH-dependent transport, remains largely unknown. This study aimed at identifying residues involved in the pH dependence of hMCT1 using a combination of amino acid-modifying reagents, site-directed mutagenesis in a Xenopus laevis oocyte expression system, and homology modeling. We showed that diethyl pyrocarbonate (DEPC), phenylglyoxal (PGO), and 4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid disodium salt (DIDS), which react with histidine, arginine, and lysine residues respectively, all inhibited hMCT1 activity. Since DEPC, PGO, and DIDS are membrane impermeable reagents, we mutated to other residues individual histidine, arginine, and lysine residues located within the extracellular regions of hMCT1. Analyses of these mutants demonstrated that except for K38, the extracellular basic residues of hMCT1 were not involved in its transport activity and pH dependence. Moreover, analyses of various mutants in which K38 was substituted for another residue and of an hMCT1 homology model focusing on the location of K38 in the three-dimensional structure delineated the mechanism of hMCT1 pH dependence. Collectively, our data indicate that K38 plays an essential role in hMCT1 transport activity. We would like to propose a mechanism whereby K38 is positioned within a hydrophobic and narrow cavity that is part of the transport pathway, and regulates pH-dependent gating of hMCT1.

Identification and characterization of the novel nuclease activity of human phospholipid scramblase 1.

BackgroundHuman phospholipid scramblase 1 (hPLSCR1) was initially identified as a Ca2+ dependent phospholipid translocator involved in disrupting membrane asymmetry. Recent reports revealed that hPLSCR1 acts as a multifunctional signaling molecule rather than functioning as scramblase. hPLSCR1 is overexpressed in a variety of tumor cells and is known to interact with a number of protein molecules implying diverse functions.ResultsIn this study, the nuclease activity of recombinant hPLSCR1 and its biochemical properties have been determined. Point mutations were generated to identify the critical region responsible for the nuclease activity. Recombinant hPLSCR1 exhibits Mg2+ dependent nuclease activity with an optimum pH and temperature of 8.5 and 37 °C respectively. Experiments with amino acid modifying reagents revealed that histidine, cysteine and arginine residues were crucial for its function. hPLSCR1 has five histidine residues and point mutations of histidine residues to alanine in hPLSCR1 resulted in 60 % loss in nuclease activity. Thus histidine residues could play a critical role in the nuclease activity of hPLSCR1.ConclusionsThis is the first report on the novel nuclease activity of the multi-functional hPLSCR1. hPLSCR1 shows a metal dependent nuclease activity which could play a role in key cellular processes that needs to be further investigated.

Three conserved histidine residues contribute to mitochondrial iron transport through mitoferrins

Iron is an essential element for almost all organisms. In eukaryotes, it is mainly used in mitochondria for the biosynthesis of iron-sulfur clusters and haem group maturation. Iron is delivered into the mitochondrion by mitoferrins, members of the MCF (mitochondrial carrier family), through an unknown mechanism. In the present study, the yeast homologues of these proteins, Mrs3p (mitochondrial RNA splicing 3) and Mrs4p, were studied by inserting them into liposomes. In this context, they could transport Fe2+ across the proteoliposome membrane, as shown using the iron chelator bathophenanthroline. A series of amino acid-modifying reagents were screened for their effects on Mrs3p-mediated iron transport. The results of the present study suggest that carboxy and imidazole groups are essential for iron transport. This was confirmed by invivo complementation assays, which demonstrated that three highly conserved histidine residues are important for Mrs3p function. These histidine residues are not conserved in other MCF members and thus they are likely to play a specific role in iron transport. A model describing how these residues help iron to transit smoothly across the carrier cavity is proposed and compared with the structural and biochemical data available for other carriers in this family.

Transport mechanisms of flavanone aglycones across Caco-2 cell monolayers and artificial PAMPA membranes

We recently reported that flavanone aglycones (hesperetin, naringenin and eriodictyol) are efficiently absorbed via proton-coupled active transport, in addition to transcellular passive diffusion, in Caco-2 cells. Here, we aimed to evaluate in detail the absorption mechanisms of these flavanones, as well as homoeriodictyol and sakuranetin. We evaluated the absorption mechanisms of the above compounds by means of in vitro studies in Caco-2 cells in parallel with an artificial membrane permeation assay (PAMPA) under pH-gradient and iso-pH conditions. Comparison of the permeability characteristics of flavanones in Caco-2 cells and in PAMPA under these conditions, as well as a consideration of the physicochemical properties, indicated that hesperetin, naringenin, eriodictyol and homoeriodictyol were efficiently transported by passive diffusion according to the pH-partition hypothesis, except in the case of sakuranetin. However, transport of all flavanones were remarkably temperature-dependent, and was significantly reduced when Caco-2 cells were treated with amino acid-modifying reagents. Our data confirm that both passive diffusion and an active transport mechanism contribute to flavanone absorption through human intestinal epithelium.

Metabolism of conjugated sterols in eggplant. Part 1. UDP-glucose : sterol glucosyltransferase.

A membrane-bound UDP-glucose : sterol glucosyltransferase from Solanum melongena (eggplant) leaves was partially purified and its specificity as well as molecular and kinetic properties were defined. Among a wide spectrum of 3-OH steroids (i.e. typical plant sterols, androstane, pregnane and cholestane derivatives, steroidal alkaloids and sapogenins) and triterpenic alcohols, the highest activity was found with 22-oxycholesterol. UDP-glucose appeared to be the best sugar donor. The enzyme preparation was also able to utilize UDP-galactose, TDP-glucose and CDP-glucose as a sugar source for sterol glucosylation, however, at distinctly lower rates. The investigated glucosyltrasferase was stimulated by 2-mercaptoethanol, Triton X-100 and negatively charged phospholipids, and inhibited in the presence of UDP, mono-, di- and triacylglycerols, divalent cations such as Zn(2+), Co(2+), high ionic strength, cholesteryl glucoside, galactoside and xyloside and some amino acid-modifying reagents (SITS, DIDS, PLP, DEPC, pCMBS, NEM, WRK and HNB). Our results suggest that unmodified residues of lysine, tryptophan, cysteine, histidine and dicarboxylic amino acids are essential for full enzymatic activity and indicate that a glutamic (or aspartic) acid residue is necessary for the binding of sugar donor, i.e. UDP-glucose in the active site of the GT-ase while histidine and cysteine residues are both important for the binding of the nucleotide-sugar as well as of the steroidal aglycone.

Purification and biochemical characterisation of a membrane-bound α-glucosidase from the parasite Entamoeba histolytica

An α-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-α- d-glucopyranoside (MUαGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 °C. Although the enzyme preferentially hydrolysed nigerose (α1,3-linked), it also cleaved kojibiose (α1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (α1,4), trehalose (α1,1) and isomaltose (α1,6). Activity on α1,3- and α1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy- d-glucose and 6-deoxy- d-glucose were strong inhibitors of activity, whereas 2-deoxy- d-glucose and other monosaccharides had no effect. Enzyme activity on MUαGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)- N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.

Essential sulfhydryl groups in the active site of castor bean ( Ricinus communis) seed acid phosphatase

In order to determine which amino acids are involved in substrate binding, castor bean seeds acid phosphatase was treated with amino acid-modifying reagents, such as phenylglyoxal (PGO), iodoacetic acid (IAA), N-bromosuccinimide (NBS), N-acetylimidazole (NAI), diethylpyrocarbonate (DEPC), N,N′-dicyclohexylcarbodiimide (DCCD) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), specific for arginine, cysteine, tryptophan, tyrosine, histidine, and aspartic and glutamic acids, respectively. Enzyme activity was determined using p-nitrophenylphosphate ( pNPP) as substrate. Enzyme inhibition was observed with IAA, NBS, EDC and DCCD. In the presence of the reaction products, p-nitrophenol ( pNP) and inorganic phosphate (Pi), which are competitive inhibitors, the enzyme was protected from inactivation by IAA, indicating involvement of the enzyme active site. The inhibition by IAA was time- and concentration-dependent, with an apparent bimolecular rate constant of 48×10 −4 M −1 s −1, and two molecules of IAA bound per active site. Our results suggest that sulfhydryl groups are essential for enzyme catalysis, and are located in or near the substrate-binding domain. Other amino acids such as tryptophan, aspartic and glutamic acids, were also important for the enzyme activity, but were probably located outside of the active site.

The Peroxisome Proliferator-induced Cytosolic Type I Acyl-CoA Thioesterase (CTE-I) Is a Serine-Histidine-Aspartic Acid α/β Hydrolase

Long-chain acyl-CoA thioesterases hydrolyze long-chain acyl-CoAs to the corresponding free fatty acid and CoASH and may therefore play important roles in regulation of lipid metabolism. We have recently cloned four members of a highly conserved acyl-CoA thioesterase multigene family expressed in cytosol (CTE-I), mitochondria (MTE-I), and peroxisomes (PTE-Ia and -Ib), all of which are regulated via the peroxisome proliferator-activated receptor alpha (Hunt, M. C., Nousiainen, S. E. B., Huttunen, M. K., Orii, K. E., Svensson, L. T., and Alexson, S. E. H. (1999) J. Biol. Chem. 274, 34317-34326). Sequence comparison revealed the presence of putative active-site serine motifs (GXSXG) in all four acyl-CoA thioesterases. In the present study we have expressed CTE-I in Escherichia coli and characterized the recombinant protein with respect to sensitivity to various amino acid reactive compounds. The recombinant CTE-I was inhibited by phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, suggesting the involvement of serine and histidine residues for the activity. Extensive sequence analysis pinpointed Ser(232), Asp(324), and His(358) as the likely components of a catalytic triad, and site-directed mutagenesis verified the importance of these residues for the catalytic activity. A S232C mutant retained about 2% of the wild type activity and incubation with (14)C-palmitoyl-CoA strongly labeled this mutant protein, in contrast to wild-type enzyme, indicating that deacylation of the acyl-enzyme intermediate becomes rate-limiting in this mutant protein. These data are discussed in relation to the structure/function of acyl-CoA thioesterases versus acyltransferases. Furthermore, kinetic characterization of recombinant CTE-I showed that this enzyme appears to be a true acyl-CoA thioesterase being highly specific for C(12)-C(20) acyl-CoAs.

Biochemical Characterization and Molecular Cloning of an α-1,2-Fucosyltransferase That Catalyzes the Last Step of Cell Wall Xyloglucan Biosynthesis in Pea

Pea microsomes contain an alpha-fucosyltransferase that incorporates fucose from GDP-fucose into xyloglucan, adding it preferentially to the 2-O-position of the galactosyl residue closest to the reducing end of the repeating subunit. This enzyme was solubilized with detergent and purified by affinity chromatography on GDP-hexanolamine-agarose followed by gel filtration. By utilizing peptide sequences obtained from the purified enzyme, a cDNA clone was isolated that encodes a 565-amino acid protein with a predicted molecular mass of 64 kDa and shows 62.3% identity to its Arabidopsis homolog. The purified transferase migrates at approximately 63 kDa by SDS-polyacrylamide gel electrophoresis but elutes from the gel filtration column as an active protein of higher molecular weight ( approximately 250 kDa), indicating that the active form is an oligomer. The enzyme is specific for xyloglucan and is inhibited by xyloglucan oligosaccharides and by the by-product GDP. The enzyme has a neutral pH optimum and does not require divalent ions. Kinetic analysis indicates that GDP-fucose and xyloglucan associate with the enzyme in a random order. N-Ethylmaleimide, a cysteine-specific modifying reagent, had little effect on activity, although several other amino acid-modifying reagents strongly inhibited activity.

The effect of amino acid modifying reagents on the activity of a (1→3)-β-glucan synthase from Italian ryegrass (Lolium multiflorum) endosperm

Abstract A series of amino acid modifying reagents were tested for their effects on the activity of the (1→3)-β-glucan synthase (EC 2.4.1.34) from endosperm of ryegrass ( Lolium multiflorum ). Of these reagents only the carbodiimide EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DEPC (diethyl pyrocarbonate), iodine, Cu 2+ , Hg 2+ , phenylmercuric nitrate, N -bromosuccinimide and 2-hydroxy-5-nitrobenzylbromide gave significant inhibition. It was concluded that carboxyl and His groups are likely to be essential for enzyme activity but the involvement of Trp residues was not excluded. Significantly, free thiols or disulfides do not appear to be necessary for enzyme activity. The kinetics of the inhibition reaction with diethyl pyrocarbonate were complex. No evidence for methoxyformylation of His was obtained by measurements of absorbance at 242 nm. EDC inhibited in a time and concentration dependent manner, with kinetics suggesting that no reversible complex between the enzyme and the EDC was formed. The apparent bimolecular rate constant was 0.029 M −1 s −1 . The order of the inactivation was 1 indicating that one molecule of EDC is bound per active site. The rate of inhibition is slowed in the presence of UDP–Glc, suggesting that the modified groups may be located in or near the substrate-binding domain. The pH dependence of the rate of inactivation indicates the presence of a critical group with a p K a of 5.7. These results are consistent with the finding that a `D,D,D(35)Q(RQ)XRW' motif is present in a number of repetitive β-glycan synthases and suggest that the critical acidic group may be an aspartate.

Essential arginine residues in isoprenylcysteine protein carboxyl methyltransferase

We used specific amino acid modifying reagents to characterize the isoprenylcysteine carboxyl methyltransferase in kidney membranes. The enzyme was inactivated by reagents specific for arginine, histidine, cysteine, and tryptophan residues. Protection by the product and inhibitor S-adenosyl-L-homocysteine was observed for arginine modification by phenylglyoxal and tryptophan modification by N-bromosuccinimide. We focused on modification by phenylglyoxal, a highly specific modifier of arginine residues. The inactivation of methyltransferase by phenylglyoxal follows pseudo-first-order kinetics and the order of the reaction, n, with respect to phenylglyoxal was 1.2. The inactivation increased with the alkalinity of the preincubation medium and was maximal at pH 10. Kinetic analysis showed that the K(m) for S-adenosyl-L-methionine is not significantly affected by treatment with phenylglyoxal but that the Vmax is reduced p-Hydroxyphenylglyoxal, a more hydrophilic derivative of phenylglyoxal, was a less potent inactivator of methyltransferase than phenylglyoxal, suggesting that arginine residues modified are in a hydrophobic environment. The methyltransferase is protected from phenylglyoxal modification by S-adenosyl-L-homocysteine but not S-adenosyl-L-methionine, sinefungin, N-acetyl-S-farnesyl-L-cysteine, or farnesylthioacetate. The arginine residue modified may thus be located either at the active site or at another additional binding site for S-adenosyl-L-homocysteine. These results indicate that arginine residues are essential for the enzymatic activity of isoprenylcysteine carboxyl methyltransferase.

Threonine diffusion and threonine transport in Corynebacterium glutamicum and their role in threonine production

Transmembrane threonine fluxes (i.e., uptake, diffusion, and carrier-mediated excretion) all contribut-ing to threonine production by a recombinant strain of Corynebacterium glutamicum, were analyzed and quantitated. A threonine-uptake carrier that transports threonine in symport with sodium ions was identified. Under production conditions (i.e., when internal threonine is high), this uptake system catalyzed predominantly threonine/threonine exchange. Threonine export via the uptake system was excluded. Threonine efflux from the cells was shown to comprise both carrier-mediated excretion and passive diffusion. The latter process was analyzed after inhibition of all carrier-mediated fluxes. Threonine diffusion was found to proceed with a first-order rate constant of 0.003 min–1 or 0.004 μl min–1 (mg dry wt.)–1, which corresponds to a permeability of 8 × 10–10 cm s–1. According to this permeability, less than 10% of the efflux observed under optimal conditions takes place via diffusion, and more than 90% must result from the activity of the excretion carrier. In addition, the excretion carrier was identified by (1) inhibition of its activity by amino acid modifying reagents and (2) its dependence on metabolic energy in the form of the membrane potential. Activity of the excretion system depended on the membrane potential, but not on the presence of sodium ions. Threonine export in antiport against protons is proposed.

Transcellular transport of benzoic acid across Caco-2 cells by a pH-dependent and carrier-mediated transport mechanism.

The pH-dependent transcellular transport of [14C]benzoic acid across a Caco-2 cell monolayer is shown to be mediated by a monocarboxylic acid-specific carrier-mediated transport system, localized on the apical membrane. Evidence for the carrier-mediated transport of benzoic acid includes (a) the significant temperature and concentration dependence, (b) the metabolic energy dependence, (c) the inhibition by unlabeled benzoic acid and other monocarboxylic acids, (d) countertransport effects on the uptake of [14C]benzoic acid, and (e) effects of a proteinase (papain) and amino acid-modifying reagents. Furthermore, since carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin significantly inhibited the transport of [14C]benzoic acid, the direct driving force for benzoic acid transport is suggested to be the inwardly directed proton gradient. From these results, together with previous observations using intestinal brush border membrane vesicles, the pH dependence of the transcellular transport of certain organic weak acids across Caco-2 cells is considered to result mainly from a proton gradient-dependent, carrier-mediated transport mechanism, rather than passive diffusion according to the pH-partition theory.

Essential amino acids involved in glucan-dependent aggregation of Streptococcus sobrinus

The active site of the glucan-binding lectin (or agglutinin) (GBL) of Streptococcus sobrinus was probed by specific amino acid modifying reagents. Reagents specific for carboxylates, imidazolium, phenolic, and lysyl residues inactivated the cell bound GBL, whereas agents specific for sulfhydryl, disulfide, and guanidinium groups had no effect on the lectin. A low molecular weight α-(1 → 6)-glucan provided partial protection against the reagents which inactivated the protein, whereas an α-(1 → 4)-glucan, incapable of complexing with the lectin, afforded no protection. A reagent specific for tryptophan, 2-hydroxy-5-nitrobenzyl bromide (HNB) did not cause a loss of GBL activity, although N-bromosuccinimide, a reagent capable of oxidizing tryptophan and less selective than HNB, was a very effective inhibitor of the glucan-dependent cellular aggregation. In the latter case, α-(1 → 6)-glucan did not protect. Hydroxylamine partially restored the loss of lectin activity due to treatment of the cells with N-acetylimidazole (highly specific for tyrosine), glycine methyl ester plus water-soluble carbodiimide (specific for carboxylates), and diethylpyrocarbonate (specific for histidine). Because the soluble form of GBL rapidly loses activity when purified, it was necessary to perform the chemical modification of the amino acid side chains employing the cell-bound form of the lectin. Because specific ligand [α-(1 → 6)-glucan] protected against the inactivation of the agglutinin by selected reagents and because lectin activity could be restored in some cases, it was possible to identify likely essential amino acid residues needed for glucan binding. The results, taken together, suggest that aspartic (and/or glutamic) acid, histidine, lysine, and tyrosine are critical amino acids responsible for agglutinin activity. Present efforts are directed to the design and synthesis of glucan analogues which may serve as affinity inactivating agents of the lectin. Such glucan derivatives may be of value in studies on the role of the lectin in cariogenesis.

Purification and Properties of Xylose Isomerase of Streptomyces coelicolor A3 (2)

AbstractThe xylose isomerase of Streptomyces coelicolor A3 (2) was purified by conventional techniques to apparent homogeneity. The relative molecular mass of the sub‐unit of the enzyme was determined as 40kDa and the enzyme is composed of 4 such sub‐units. Significantly, the enzyme is optimally active at pH 7.0 and at 70°C, and requires only magnesium as cofactor. Presence of magnesium considerably minimizes inhibition of the enzyme by calcium. These considerations suggest that the present enzyme is more ideal for application in high fructose syrup process. The enzyme has Km = 0.041 M for xylose and 0.2 M for glucose with the corresponding Vmax values being 58.4 and 9.09 mol. min−1 mg−1. Among the various amino acid modifying reagents, acetic anhydride and ethyl dimethyl amino propyl carbodiimide showed the most pronounced effects of reduction and enhancement of catalysis respectively.

O-acetylation and de-O-acetylation of sialic acids: O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues—a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.

Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III).

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.

THE DIAPAUSE FACTOR FROM THE SILKWORM, BOMBYX MORI L.: PURIFICATION AND INACTIVATION EXPERIMENTS.

The diapause factor, which is responsible for the induction of èmbryonic dia pause in the silkworm (Bombyx mori L.), has been partially purified from the extract of adult heads by means of protein purification procedures, including the use of gel filtration of Sephadex, column chromatography on Dowex 1, isoelectric focusing and phenol extraction. Two species of the diapause factor could be recognized in respect to their molecular weight. They were separated by Sephadex G-25 and their molecular weights were estimated to be about 2,000 and 5,000 from the gel filtration results. The smaller species was purified about 90-fold in specific activity, and its isoelectric point was determined by isoelectric focusing to be at about pH 4.5. The biological activity of the partially purified principle could be abolished by incubation with several proteolytic enzymes (trypsin, α-chymotrypsin and pronase), or by treatment with amino acid-modifying reagents such as tyrosinase, N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide, but was not affected by incubation with neuraminidase, cyanogen bromide or photooxidation in the presence of methylene blue.

Glutamine synthetase from rice plant roots

Glutamine synthetase [ l-glutamate: ammonia ligase (ADP), EC 6.3.1.2] was partially purified from rice plant roots grown on ammonium medium, and some properties were investigated. The apparent K m values of the various substrates and cofactors were determined. Lineweaver-Burk plots gave Michaelis constants ( K m ) of of 3.75 m m with l-glutamate, 0.44 m m with ATP, and 0.4 m m with NH 2OH. Michaelis constants for Mg 2+ and Mn 2+ appeared to differ; K m for Mg 2+ was approximately one order of magnitude higher than for Mn 2+. Metals, including Cu 2+, Hg 2+, Cd 2+, Zn 2+, Ca 2+, and Ni 2+, strongly inhibited the enzyme activity. Contrary to the B. subtilis enzyme, Fe 2+ and Fe 3+ strongly inhibited while Co 2+ slightly stimulated. Preincubation of enzyme with phenylmercuric acetate or p-hydroxymercuriphenylsulfonic acid (1 m m) as thiol reagent resulted in marked inhibition. Cysteine reactivated the enzyme activity which had been inactivated by preincubation with phenylmercuric acetate of HgCl 2. The enzyme activity was essentially destroyed by amino acid modifying reagents such as N-bromosuccinimide and N-ethylmaleimide. α-Keto-glutarate and citrate increased the enzyme activity by about 1.5-fold as in agreement with the data obtained in rat liver enzyme. Pyruvate and glyoxylate (20 m m) significantly inhibited. Among the nucleotides tested, AMP, ADP, GTP, and IDP gave substantial inhibition dependent on their physiological concentrations. AMP and ADP showed a competitive inhibition with respect to ATP, while GTP a strong uncompetitive inhibition against ATP and a mixed-type inhibition with regard to glutamate. Moreover, combined effects of these nucleotides resulted in cumulative inhibition. In contrast to the B. subtilis enzyme, glutamine (20 m m) slightly stimulated rice plant enzyme. l-Alanine, l-aspartate, l-asparagine, l-glycine, and l-histidine did not affect the activity unlike the microbial enzymes. These results are discussed in relation to the regulatory properties of glutamine synthetase in rice plant roots.