Effect of divalent metal ions on the synthesis of oligosaccharide side chains of Neurospora crassa glycoproteins

Neurospora crassa membrane preparations incorporated mannose from GDP-mannose-[¹⁴C] in the presence of Mg²⁺ into a polyprenol lipid and side chains of protein acceptor(s), which are labile on hydrolysis in weak base. The addition of Mn²⁺ to the reaction mixtures does not affect mannosyl lipid synthesis but it stimulates the transfer of mannose to larger oligosaccharide chains resistant to β-elimination and the transfer of a second mannosyl unit to form an O-glycosidically linked mannobiosyl side chain. Incubation of particulate preparations with polyprenol-mannose-[¹⁴C] in the presence of Mg²⁺ and Mn²⁺ also results in the transfer of a single mannose to the protein. When non-radioactive GDP-mannose is added to this reaction mixture, however, β-elimination yields mannobiose. The mannobiose is labeled in the reducing sugar only. These results indicate that the first mannose of this mannobiosyl side chain is transferred via a lipid intermediate, but the second mannose is transferred directly from GDP-mannose. In the presence of Mg²⁺ and Mn²⁺, mannose apparently is also transferred from polyprenol-mannose-[¹⁴C] to side chains which are resistant to hydrolysis.     

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L: Involvement of Lipid-linked Intermediates

Particulate preparations from developing cotyledons of Pisum sativum L. cv. Burpeeana catalyze glycosyl transfer from UDP-[¹⁴C]N-acetylglucosamine and GDP-[¹⁴C]mannose. Radioactivity is transferred to lipid components soluble in chloroform-methanol (2:1) and chloroform-methanol-water (1:1:0.3) and into a water-insoluble and lipid-free residue.     

Metabolism of tyramine and feruloyltyramine in TMV inoculated leaves of Nicotiana tabacum

TMV inoculation is known to stimulate tyramine N-feruloyl-CoA transferase activity in Nicotiana tabacum cv Xanthi n.c. leaves during the hypersensitive reaction. When [2-¹⁴C]-tyramine is fed for 2 hr to TMV inoculated leaf discs or detached leaves, ca 1 % of the supplied radioactivity is integrated into cinnamoyl-, p-coumaroyl- and feruloyltyramine and up to 14 % is integrated into the cell wall residue. [2-¹⁴C]-tyramine can only be partially released from this residue by acid hydrolysis. After nitrobenzene oxidation, 97 % of the radioactivity found in the cell walls is made soluble but only 13 % is recovered in p-hydroxybenzaldehyde. Feruloyltyramine is very rapidly metabolised, ca 20 % of the administrated radioactivity is found after 2 hr feeding in unindentified methanoi soluble metabolites. Acid hydrolysis of the cell wall fraction, which hydrolyses the amide bond of feruloyltyramine, releases labelled tyramine, while radioactivity is still detected in the acid insoluble residue. Label from [¹⁴C]-feruloyltyramine is integrated into this residue more quickly than from free [2-¹⁴C]-tyramine.     

Effect of Liposomes Containing Various Divalent Cations on the Release of Acetylcholine from Synaptosomes

Abstract: The effect of increasing the cytoplasmic levels of various divalent cations on the release of [³H]acetylcholine ([³H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [³H]choline were incubated with liposomes containing Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Sr²⁺, or Ba²⁺. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [³H]ACh and [³H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg²⁺, Mn²⁺, or Co²⁺ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K⁺/Na⁺. Synaptosomes incubated with liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [³H]ACh. Liposomes containing either Ca²⁺ or Sr²⁺ were equally effective in promoting the release of [³H]ACh from synaptosomes, whereas liposomes containing Ba²⁺ were 2.5 times more effective in promoting the release of [³H]ACh than were liposomes containing either Ca²⁺ or Sr²⁺. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [³H]ACh caused by liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.     

The enzymatic conversion of cis-(14C)phytofluene, trans-(14C)phytofluene, and trans-zeta-(14C)carotene to more unsaturated acyclic, monocyclic, and dicyclic carotenes by a cell-free preparation of red tomato fruits

This paper reports the conversion of cis-[¹⁴C]phytofluene to trans-[¹⁴C|phytofluene and the conversion of the latter compound to trans-ζ-[¹⁴C]carotene by a soluble enzyme system obtained from the plastids of red tomato fruits. Each of these radioactive compounds was also converted to labeled neurosporene, lycopenc, α-carotene, and β-carotene by the same enzyme system. The incorporation of each substrate into more unsaturated carotenes was carried out under nitrogen at pH 7.5–8.2 (borate buffer), at 25 °C in the dark.Proof of the formation of the above carotenes from each of the three radioactive substrates was demonstrated by cochromatography with authentic nonradioactive carotenes on an alumina chromatographic column. A close correspondence between radioactivity and light absorbance for each carotene was observed. Confirmation of these conversions was achieved by cochromatography with authentic samples on thinlayer plates. Final proof for the formation of the acyclic and cyclic carotenes from the above radioactive substrates was obtained by gas-liquid chromatography of the hydrogenated products. Coincidence between mass and radioactivity was observed.Maximum conversion of cis- and trans-phytofluenes to more unsaturated carotenes by the red tomato fruit enzyme system appears to be dependent upon the presence of NADP⁺, FAD, and Tween 80. The formation of the carotenes is also increased in the presence of Mg²⁺ or Mn²⁺ ions.     

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP-N-acetyl-[¹⁴C]glucosamine in the presence of Mg²⁺ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl₃CH₃OHH₂O (10:10:3) extract after incubating with only UDP-N-acetyl-[¹⁴C] glucosamine, Mg²⁺, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[¹⁴C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl₃CH₃OHH₂O (10:10:3). The [GlcNAc-¹⁴C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-¹⁴C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[¹⁴C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[¹⁴C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[¹⁴C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.     

Biosynthesis of O-acyl ethanediol phosphorylcholine in a cell-free system from rat liver.

1-$\text{0}$-Hexadecanoyl [U-¹⁴C]ethanediol can serve as substrate in the formation of 1-$\text{0}$-hexadecanoyl ethanediol 2-phosphorylcholine by particulate cell-free preparations from rat liver. Catalytic activity is largely associated with the microsomal fraction. The reaction requires CDPcholine and Mg⁺⁺. Phosphatidylcholine cannot substitute for CDPcholine, but Mn⁺⁺ is almost as effective as Mg⁺⁺. Ca⁺⁺ inhibits the reaction. The acyl ethanediol phosphorylcholine produced was identified by repeated cochromotography with authentic diol phospholipid to constant specific radioactivity, and by enzymatic and chemical degradations.     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Altering the Divalent Metal Ion Preference of RNase E

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg²⁺ and Mn²⁺ will support significant rates of activity in vitro against natural RNAs, with Mn²⁺ being preferred. Both Mg²⁺ and Mn²⁺ also support cleavage of an oligonucleotide substrate with similar kinetic parameters for both ions. Salts of Ni²⁺ and Zn²⁺ permitted low levels of activity, while Ca²⁺, Co³⁺, Cu²⁺, and Fe²⁺ did not. A mutation to one of the residues known to chelate Mg²⁺, D346C, led to almost complete loss of activity dependent on Mg²⁺; however, the activity of the mutant enzyme was fully restored by the presence of Mn²⁺ with kinetic parameters fully equivalent to those of wild-type enzyme. A similar mutation to the other chelating residue, D303C, resulted in nearly full loss of activity regardless of metal ion. The properties of RNase E D346C enabled a test of the ionic requirements of RNase E in vivo. Plasmid shuffling experiments showed that both rneD303C (i.e., the rne gene encoding a D-to-C change at position 303) and rneD346C were inviable whether or not the selection medium was supplied with MnSO₄, implying that RNase E relies on Mg²⁺ exclusively in vivo.     

A dolichol-linked trisaccharide from central nervous tissue: structure and biosynthesis.

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[¹⁴C]glucosamine from UDP-N-acetyl[¹⁴C]glucosamine into N-acetyl[¹⁴C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[¹⁴C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [¹⁴C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[¹⁴C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [¹⁴C]trisaccharide lipid. The intact [¹⁴C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [¹⁴C]trisaccharide is converted to N,N′-diacetyl[¹⁴C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [¹⁴C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[¹⁴C] glucosaminylpyrophosphoryldolichol to form [¹⁴C]disaccharide lipid. The biosynthesis of [¹⁴C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [¹⁴C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [¹⁴C] trisaccharide lipid. The formation of the [¹⁴C]trisaccharide lipid from exogenous [¹⁴C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg²⁺, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.     

Regulation of rat-kidney cortex fructose-1,6-bisphosphatase activity. I. Effects of fructose-2,6-bisphosphate and divalent cations

• 1.1. The native rat-kidney cortex Fructose-1,6-BPase is differentially regulated by Mg²⁺ and Mn²⁺.
• 2.2. Mg²⁺ binding to the enzyme is hyperbolic and large concentrations of the cation are non-inhibitory.
• 3.3. Mn²⁺ produces a 10-fold rise in V_max higher than Mg²⁺. [Mn²⁺]_0.5 is much larger than [Mg²⁺]_0.5. At elevated [Mn²⁺] inhibition is observed.
• 4.4. Mg²⁺ and Mn²⁺ produce antagonistic effects on the inhibition of the enzyme by high substrate.
• 5.5. Fru-2,6-P₂ inhibits the enzyme by rising the S_0.5 and favouring a sigmoidal kinetics.
• 6.6. The inhibition by Fru-2,6-P₂ is released by Mg²⁺ and more powerfully by Mn²⁺ increasing the I_0.5.

     

The regulatory role of Mg2+ on rabbit skeletal muscle phosphorylase kinase

The stimulation of phosphorylase kinase by Mg²⁺ was studied. Both the nonactivated and activated kinases are stimulated by Mg²⁺ at concentrations that are 100- to 200-fold greater than ATP. This stimulation is observed at both pH 6.8 and 8.2 and results in a 10-fold increase in the activity of the nonactivated kinase. Mg²⁺ stimulation is additive with that observed by calmodulin. Both the Ca²⁺-dependent and -independent activities of the kinase are stimulated by high [Mg²⁺]. Kinetically this stimulation can be explained by a decrease in the K_m for both phosphorylase b and ATP or an increase in V. The pH $\text{6.8}\text{8.2}$ ratio (0.06) is unaffected by [Mg²⁺] between 5 and 20 mm, but increases when [Mg²⁺] is less than 5 mm or greater than 20 mm. The stimulation by high [Mg²⁺] is explained by a direct effect of this cation on the kinase molecule rather than on its protein substrate, phosphorylase. This activating effect of high [Mg²⁺] does not result in any permanent change in the kinase molecule and can be readily reversed by diluting [Mg²⁺] to a low value.     

Glucolipid Synthesis in Acanthamoeba castellanii*

SYNOPSIS. Cell-free preparations of Acanthamoeba castellanii trophozoites transfer glucose from UDP-[U-¹⁴C]glucose to a chloroform-soluble form. This radioactive material has been isolated by thin-layer chromatography; it contains an alkali-labile and an alkali-stable (unsaponifiable) component. Treatment of the enzymic product with 0.1 N KOH for 15 min at 0 C or 20 C releases radioactivity into the aqueous phase as glucose. During this treatment, 30–60% of the original glycolipid remains chloroform-soluble. It is considered to be an alkali-stable glycolipid because no further loss of radioactivity occurs during an additional 45-min of treatment with 0.1 N KOH. During incubation with 0.1 N HCI at 100 C glucose is released quantitatively from both the untreated glycolipid and the alkali-stable glycolipid with a half-time of 6 min. Glycolipid formation is inhibited by UDP and is reversible; extracts catalyze the formation of UDP-glucose from the alkali-stable glucolipid and UDP. The chemical and physical properties of the alkali-stable glycolipid are consistent with a glucosyl phosphoryl polyprenol structure. Extracts prepared from cysts catalyze the formation of glycolipids aiso, but the glucosyltransferase activity/cell decreases during the course of encystment. Radioactivity is incorporated into the fraction insoluble in chloroform-methanol-water (1:1:1:) during these incubations when UDP-[U-¹⁴C]glucose or [¹⁴C]glycolipid is the substrate.     

Studies on isoprenoid biosynthesis with bacterial intact cells

For the study on the regulation of isoprenoid biosynthesis with intact cells, some strains of bacteria capable of growing on mevalonate as a sole carbon source were isolated from soil. Many of them incorporated [¹⁴C]-mevalonate, [¹⁴C]isopentenyl- and [¹⁴C]farnesyl pyrophosphates into the cells. However, radioactivity was found in their degradation products but not in isoprenoids. Addition of [¹⁴C]isopentenyl pyrophosphate, farnesyl pyrophosphate and Mg²⁺ ions in combination to the culture of a strain of Arthrobacter gave rise to ¹⁴C-incorporation into isoprenoids. Radioactivity was found in polyprenol, its pyrophosphate, monophosphate and fatty acid esters. The reactions of isopentenyl- and farnesyl pyrophosphates syntheses seemed to be rate-limiting steps.     

Regulation of smooth muscle phosphatase-II by divalent cations

Summary Smooth Muscle Phosphatases II (SMP-I1) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg²⁺ and Mn²⁺ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg²⁺ is 1.5–2.5-fold higher than the maximal Mn²⁺ activation. However, the latter is achieved at a lower concentration than the maximal Mg²⁺-activation. Furthermore, at low cation concentrations ( 2 mM), the Mn²⁺-activated activity is higher than the Mg²⁺-activated activity. In the presence of both cations, the effect of Mn²⁺ predominates suggesting that the affinity of the enzyme for Mn²⁺ is greater than for Mg²⁺. In contrast to Mg²⁺ and Mn²⁺, Ca²⁺ does not activate SMP-II but it was observed to antagonize the effects of Mg²⁺ and Mn²⁺. Ca²⁺ acts as a competitive inhibitor of Mg²⁺. However, the inhibitory effect at high Ca²⁺ concentrations is not completely reversed by increasing the Mg²⁺ concentration. Mn²⁺ activation is also inhibited by Ca²⁺ but to a lesser extent. Ca²⁺ cannot completely inhibit Mn²⁺-activation suggesting that SMP-I1 has greater affinity for Mn²⁺ than for Ca²⁺. The finding that Ca²⁺ inhibits the activation of SMP-II raises the possibility that Ca²⁺ may be a regulator of SMP-II in vivo.Abbreviations SMP-II Smooth Muscle Phosphatase-II - MOPS 3-[N-Morpholine]propane Sulfonic Acid - PLC Phosphorylated Myosin Light Chains     

Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate

The properties of the tight and specific binding of 2-C-carboxy-d-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO₂ and Mg²⁺, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of [¹⁴C]CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported (S Johal, BE Partridge, R Chollet [1985] J Biol Chem 260: 9894-9904). The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg²⁺. Addition of [¹⁴C]CABP and EDTA stopped binding of Mg²⁺ and allowed tight binding of the radiolabel only to sites which were CO₂/Mg²⁺-activated at that moment. This approach estimated the amount of CO₂/Mg²⁺-activated sites in the presence of inactive sites and carbamylated sites lacking Mg²⁺. The rate of CO₂ fixation was proportional to the CO₂/Mg²⁺-activated sites. During light-dependent CO₂ fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg²⁺ and CO₂. Lysis of chloroplasts in media with [¹⁴C]CABP plus EDTA estimated those carbamylated sites having Mg²⁺. The loss of Rubisco activation during illumination was partially due to the lack of Mg²⁺ to stabilize the carbamylated sites.     

Characterization of mannosyl transferases during the pupal instar of Stomoxys calcitrans (L)

Particulate fractions (10,000g) from pupae of Stomoxys calcitrans transfer [¹⁴C]-mannose from GDP-[¹⁴C]-mannose to dolichol monophosphate and proteins. Production of the mannosyl lipid was inhibited by Mn²⁺, UDP, GMP, GDP, and EDTA. The insect growth regulator diflubenzuron had no effect on mannosyl transferase activity. Dolichol monophosphate and Mg²⁺ stimulated mannosyl transferase activity. The mannosyl lipid product was identified as mannosyl-phosphoryl-dolichol (Man-P-Dol). The apparent K_m and V_max values for the formation of Man-P-Dol using GDP-[¹⁴C]-Man while holding dolichol phosphate constant were 2.4 ± 0.9 μM and 9.4 ± 2.3 pmol Man-P-Dol·min^?1·mg^?1 protein, respectively. The apparent K_m and V_max values using dólichol phosphate while holding GDP-Man constant were 2.2 ± 1.2 μM and 18.5 ± 1.7 pmol Man-P-Dol·min^?1·mg^?1 protein.     

Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides: II. Glucosylated units

Pig thyroid rough microsomes catalyzed the transfer of glucose from UDP-[¹⁴C]Glc to glycolipids extractable with chloroform/methanol, glycolipids extractable with a water-saturated chloroform/methanol and to a residual material. Kinetics of labeling were compatible with a precursor-product relationship between the second type of glycolipid and residuals.The [¹⁴C] Glc-glycolipids soluble in CHCl₃/CH₃OH/H₂O, 10 : 10 : 3, behaved on DEAE-cellulose mainly as pyrophospho derivatives, with some less acidic radioactivity, probably $\text{dolichol}\text{-P-[}{}^{14}\text{C}\text{]}\text{Glc}$. Their saccharide moieties released by mild acid appeared polydisperse on paper chromatography, a part of them being estimated larger than a nonasaccharide marker GlcNAc-[Man]₈. The ¹⁴C-labeled glucosylated glycoproteins have represented all the considerable polymeric label remaining after lipid extraction. Their pronase glycopeptides were submitted to a differential reductive alkaline hydrolysis and it was concluded that their [¹⁴C] glucose belongs mainly to N-glycosically linked units. On gel filtration, the released saccharides exhibited an average size of nine monosaccharide units (from six to twelve with a relatively high proportion of material containing more than nine sugars).In a [¹⁴C] Glc-microsomal extract, 29% of the non-lipid radioactivity was found immunoreactive with an antiserum to pig thyroglobulin.     

Studies on the metabolism of ATP by isolated bacterial membranes: solubilization and phosphorylation of a protein component of the diglyceride kinase system

A soluble fraction, obtained by extracting E. coli cytoplasmic membrane vesicles with water, transfers radioactivity from [γ-³²P]ATP to a protein present in this soluble fraction. The formation of the [³²P]phosphoprotein appears to be reversible. Thus the protein can transfer its ³²P to ADP to form [³²P]ATP, and the phosphate on the protein can exchange with the phosphate of ATP. Preliminary evidence indicates that the phosphate moiety is linked to a histidine residue of the protein. The Mn²⁺ and ATP dependencies of [³²P]phosphoprotein formation are almost identical to the diglyceride kinase reaction previously reported in intact membrane vesicles. Although indirect evidence supports the involvement of the phosphoprotein in the diglyceride kinase reaction, the soluble fraction catalyzes only a slow formation of [³²P]phosphatidie acid from [γ-³²P]ATP and α,β-diglyceride.     

The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding

Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB_V superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn²⁺ or Mg²⁺ cations in a dosage-dependent manner. However, whereas Mn²⁺ strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg²⁺. Furthermore, MobMN199 exhibited a high affinity binding for Mn²⁺ but not for Mg²⁺. We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein–DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn²⁺ during pMV158 transfer.