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A series of bacterial cell wall glycopeptides of low molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharide-peptides GlcNAc-MurNAc- L-Ala-D-Glu(A2pm) (C5) and GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) (C6) as well as the monosaccharide-peptide MurNAc-L-Ala-D-Glu(A2pm) were found to be good competitive in- hibitors (with similar Ki values) whereas the disaccharide-pentapeptide GlcNAcMurNAc-L-Ala-D Glu-Gly [This symbol cannot be represented into ASCII Text]-L-Lys-D-Ala was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharide-peptide C6. No changes were seen in the circular dichroism spectra (200–250 nm) or fluorescence emission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAc-L-Ala-D-Glu(A2pm). [ABSTRACT FROM AUTHOR]

The primary structure of the major parvalbumin (pI = 4.45) from the cartilaginous fish Raja clavata has been determined. The amino acid sequence was deduced by the analysis of peptides derived from tryptic digestion of the oxidized protein. These peptides were aligned by comparison with (a) overlapping peptides produced by limited tryptic and chymotryptic digestion, and (b) by comparison with the known structures of other fish parvalbumins. The molecular evolution of thornback ray parvalbumin is briefly discussed. [ABSTRACT FROM AUTHOR]

We have studied the ribonucleosidediphosphate reductase of a deoxyuridine-requiring mutant LD195 of Escherichia coli K. This enzyme was purified 50 fold from the mutant and a control strain. In all steps of the purification the mutant strain yielded normal activity of the Bi subunit of the enzyme but approximately 10 % of the wild-type activity of the B2 subunit The activity is similarly reduced for all three substrates tested CDP, UDP, and GDP. The mutation appears to affect the physical structure of the B2 subunit, since addition of high concentrations of sodium acetate can restore enzymatic activity to the partially purified B2 subunit from the mutant strain. In contrast, the same concentrations strongly inhibit the wild- type B2 subunit. Although the mutant has a requirement of deoxyuridine at 42 °C but not at 30 °C (as shown in an accompanying paper), mutant protein B2 is not more thermo-labile than wild-type protein B2 This requirement therefore probably reflects an increased demand of deoxyribonucleotides at higher temperatures. Hydroxyurea inhibits mutant and wild-type ribonucleosidediphosphate reductase to the same extent in vitro. In spite of this, hydroxyurea inhibits growth of the mutant strain at a 20 fold lower concentration than that required for comparable inhibition of growth of the parent. This is explained by the greatly decreased protein B2 activity of the mutant. [ABSTRACT FROM AUTHOR]

Previous work defined several families of secondary active transporters, including the prokaryotic small multidrug resistance (SMR) and rhamnose transporter (RhaT) families as well as the eukaryotic organellar triose phosphate transporter (TPT) and nucleotide-sugar transporter (NST) families. We show that these families as well as several other previously unrecognized families of established or putative secondary active transporters comprise a large ubiquitous superfamily found in bacteria, archaea and eukaryotes. We have designated it the drug/metabolite transporter (DMT) superfamily (transporter classification number 2.A.7) and have shown that it consists of 14 phylogenetic families, five of which include no functionally well-characterized members. The largest family in the DMT superfamily, the drug/metabolite exporter (DME) family, consists of over 100 sequenced members, several of which have been implicated in metabolite export. Each DMT family consists of proteins with a distinctive topology: four, five, nine or 10 putative transmembrane α helical spanners (TMSs) per polypeptide chain. The five TMS proteins include an N-terminal TMS lacking the four TMS proteins. The full-length proteins of 10 putative TMSs apparently arose by intragenic duplication of an element encoding a primordial five-TMS polypeptide. Sequenced members of the 14 families are tabulated and phylogenetic trees for all the families are presented. Sequence and topological analyses allow structural and functional predictions. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (Cdks) play a central role in the regulation of the eukaryotic cell cycle. A novel gene encoding a Cdk-like protein, Pjmrk, has been isolated from the human malaria parasite Plasmodium falciparum. The gene has no introns and comprises an open reading frame encoding a protein of 324 amino acids with a predicted molecular mass of 38 kDa. Database searches revealed a striking simi larity to the Cdk subfamily with the highest similarity to human MO15 (Cdk7). The overall sequence of Pfmrk shares 62% similarity and 46% identity with human MOI5, in comparison to the 49–58% similarity and 34–43% identity with other human Cdks. Pfmrk contains two unique inserts: one consisting of 5 amino acids just before the cyclin-binding motif and the other composed of 13 amino acids within the T-loop equivalent region. Southern blots of genomic DNA digests and chromosomal separations showed that Pfmrk is a single copy gene conserved between several parasite strains and is located on chromosome 10. A 2500-nucleotide transcript of this gene is expressed predominantly in the sexual blood stages (gametocytes). suggesting that Pfmrk may be involved in sexual stage development. [ABSTRACT FROM AUTHOR]

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)-binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherchia coli were overexpressed and purified. Nucleoude binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd = 32 μM and 120 μM). respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers and at pH 7.5 in sodium phosphate buffer, NAD+hound with lower affinity (Kd = 300 μM). NADPH bound only very weakly (Kd1> 1 mM). Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M & Hatefi, Y. (1993) J. Biol. Chem, 268, 1781-17877 ] found that mitochondrial transhydrogenase, and a proteolytically derrived domain I fragment from that exnzyme, bound one NADH per dimer. They suggested that this result implied half-of-the-sites reactivity for the interaction between the nucleotide ligand and the protein. However our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I protein using equilibrum dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monochondrial transhydrogenases are discussed. [ABSTRACT FROM AUTHOR]

A cytosolic 21-kDa protein isolated from bovine brain was demonstrated to bind hydrophobic ligands, particularly phosphatidylethanolamine. It was encountered in several tissues and species; however, its accurate function remained partially unknown. In order to obtain information from its structural features, we built a molecular model which revealed it to possess a nucleotide-binding site. In the present research, we describe the affinity of the bovine brain 21-kDa protein for nucleotides, and its association with cytosolic proteins, small GTP-binding proteins and lipid droplets. Our results suggest that, through its association with small GTP-binding proteins, the 21-kDa protein is implicated in signal mechanisms during cell growth and maturation. [ABSTRACT FROM AUTHOR]

From an analysis of current data on 16 protein structures with defined nucleotide-binding sites consensus motifs were determined for the peptide segments that form such nucleotide-binding sites. This was done by using the actual residues shown to contact ligands in the different protein structures, plus an additional 50 sequences for various kinases. Three peptide segments are commonly required to form the binding site for ATP or GTE Binding motif Kinase-1a is found in almost all sequences examined, and functions in binding the phosphates of the ligand. Variant versions, comparable to Kinase-1 a, are found in a subset of proteins and appear to be related to unique functions of those enzymes. Motif Kinase-2 contains the conserved aspartate that coordinates the metal ion on Mg-ATP. Motif Kinase-3 occurs in at least four versions, and functions in binding the purine base or the pentose. Two protein structures show ATP-binding at a separate regulatory site, formed by the motifs Regulatory-1 and Regulatory-2. Structures for adenylate kinase and guanylate kinase show three different sequence motifs that form the binding site for a nucleoside monophosphate (NMP) NMP-1 and NMP-2 bind to the pentose and phosphate of the bound ligand. NMP-1 is found in almost all the kinases that phosphorylate AMP, CMP, GME dTMP, or UMP NMP-3a is found in kinases for AMP, GMP and UMP, while NMP-3b binds only GMP. For the binding of NTPs, three distinct types of nueleotide-binding fold structures have been described. Each structure is associated with a particular function (e.g. transfer of the γ-phosphate, or of the adenylate to an acceptor) arid also with a particular spatial arrangement of the three Kinase segments evident in the linear sequence for the protein. [ABSTRACT FROM AUTHOR]

cAMP strongly stimulates the activity of the human prolactin (hPRL) promoter. We have previously shown that two types of cis-element are required for this cAMP regulation; binding sites for the pituitary-specific factor Pit-1, and the sequence spanning nucleotides -115 to -85 (named sequence A). Sequence A contains the TGACG motif found in the consensus sequence of the cAMP- responsive element (CRE). In this study, we show that a mutation in the TGACG motif of sequence A strongly reduces not only the cAMP regulation but also the Ca2+ regulation and basal activity of the hPRL promoter. Furthermore, gel-shift assays indicate that the mutation prevents binding of a ubiquitous factor which is not the CRE-binding protein. Southwestern experiments suggest that this ubiquitous factor's molecular mass is approximately 100 kDa. We conclude that binding of a 100-kDa ubiquitous factor to sequence A is required for full basal and hormonal regulation of hPRL- promoter activity. [ABSTRACT FROM AUTHOR]

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB- encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182–191 ). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coil is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coil using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays. [ABSTRACT FROM AUTHOR]

The nucleotide sequence encoding the chloroplast enzyme, sedoheptulose-1,7-bisphosphatase [Sed(1,7)P2ase], was obtained from wheat cDNA and genomic clones. The transcribed region of the Sed(1,7)P2ase gene has eight exons (72–507 bp) and seven introns (85–626 bp) and encodes a precursor polypeptide of 393 amino acids. Comparison of the deduced amino acid sequence of Sed(1,7)P2ase with those of fructose-1,6-bisphosphatase (Fru(1,6)P2ase] enzymes from a variety of sources reveals 19% identity, rising to 42% if conservative changes are considered. Most importantly, the amino acid residues which form the active site of Fru(1,6)P2ase are highly conserved in the Sed(1,7)P2ase molecule, indicating a common catalytic mechanism. Interestingly, although the activities of both Sed(1,7)P2ase and chloroplast Fru(1,6)P2ase are modulated by light via the thioredoxin system, the amino acid sequence motif identified as having a role in this regulation in chloroplast Fru(1,6)P2ase is not found in the Sed(1,7)P2ase enzyme. [ABSTRACT FROM AUTHOR]

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7- kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the Agt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease. [ABSTRACT FROM AUTHOR]

Mersalyl, 5,5′-dithio-bis(2-nitrobenzoate) (Nbs2) and fluorescent Thiolyte DB react with SH groups in the H+ channel (SHC) of the uncoupling protein of brown adipose tissue mitochondria, as inferred from their inhibition of H+ transport. C- transport by the uncoupling protein was unaffected. Using these modifiers and N-ethylmaleimide (MalNEt), distinct SH groups (SHB) in the purine nucleotide binding site were identified. Nbs2 reacts more readily with the SHB than with the SHC groups, but mersalyl and Thiolyte DB are more reactive with the SHC groups. MalNEt reacts exclusively with the SHB. GDP inhibition is fully prevented after sufficient modification of the SHB. Pretreatment with p-diazobenzenesulfonate (N2PhSO2) suppresses only 20–25% of fluorescence of Thiolyte-DB-labeled uncoupling protein on SDS/PAGE gels, while MalNEt suppresses 66% and Nbs2 80–90%. Since N2PhSO2 also affects the GDP binding site, these results demonstrate that the reactive residue is not identical with the SHB. [ABSTRACT FROM AUTHOR]

cAMP receptor protein from Escherichia coli has been purified on a large scale. Analogues of cAMP modified on the 6-NH2 group of the adenosine ring, the ribose 2'OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of cAMP. More extensive modification produces weaker binding. Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2'OH position remains accessible to solvent. The 2-NH2 group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent. [ABSTRACT FROM AUTHOR]

Soluble chloroplast coupling factor 1 (CF1) and the ATP synthase complex, under uncoupled conditions, can form bound ATP from tightly bound ADP and medium Pi. This partial reaction is a powerful probe of the mechanism of ATP synthesis. During our study of the synthesis of bound ATP by CF1 other enzyme activities, which generate [32P]nucleotides from 32Pi, were characterized and controlled. Two enzymes present at significant levels in the preparations are polynucleotide phosphorylase and adenylate kinase. Polynucleotide phosphorylase (PNPase) was found both in thylakoid and CF1 preparations and catalyzed the formation of [β-32P]ADP via its Pi ↔ ADP exchange activity. The formation of [32P]ADP during net photophosphorylation is attributable to adenylate kinase action on the [32P]ATP formed since hexokinase and glucose effectively block its production. In addition, PNPase also degraded RNA present in thylakoid preparations yielding all four [32P]nucleoside diphosphates. PNPase was also shown to catalyze a Pi ↔ ATP exchange that is dependent on RNA primers and other cofactors. [ABSTRACT FROM AUTHOR]

The replication of M13 single-stranded DNA by the 9S DNA polymerase α from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single- stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase α contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA. [ABSTRACT FROM AUTHOR]

The microsomal preparation from the lactating bovine mammary tissue was solubilized by treatment with nonionic detergent, NP-40, at a protein/detergent ratio of 1.5:1 and a detergent concentration of 0.5%. Following centrifugation at 147000 × g for 120 rnin, the supernatant fraction was incubated with labeled sugar nucleotides, GDP-Man and UDP-GlcNAc. It was found to synthesize a series of lipid-linked saccharides up to (Man)5- (GlcNAc)2. The solubilized glycosyltransferases retained up to about 60% of the activity after two weeks of storage at 4°C. The biosynthesis of glycolipids was stimulated by a mixture of lipids obtained by extracting the mammary microsomes with CHCl3/CH3OH (2:1). A labeled lipid-linked tetrasaccharide of the structure Man&alpha1→3ManΒ→GlcNAcΒGlcNAc was isolated by labeling baby hamster kidney cells with [2-3H]mannose under conditions of glucose starvation followed by extraction of the cells with CHCl3/CH3OH (2:1) and separation of the lipids by hiigh-performance liquid chromatography. When this lipid-linked tetrasaccharide was incubated with the solubilized bovine mammary microsomes and GDP-Man, it was elongated to a lipid-linked heptasaccharide having the structure Manα1→2Manα1&rar;2Manα1→3(Manα1→6)ManΒ→GlcNAcΒ→GIcNAc. The kinetics of the elongation reaction also revealed the intermediary formation of smaller amounts of lipid-linked pentasaccharide and hexasaccharide. The elongation reaction did not require any divalent metal ion and had a broad pH optimum between 6.8 and 7.6. The lack of inhibition of the elongation reaction by EDTA or amphomycin support earlier studies that GDP-Man rather than mannosylphosphoryldolichol, is the direct donor of mannosyl residues for the biosynthesis of glycolipids up to (Man)5(GlcNAc)2. Mannosylphosphorylretinol was ineffective as rnannosyl donor for the elongation reaction. [ABSTRACT FROM AUTHOR]

Guanosine 3',5'-rnonophosphate (3',5'GMP) activates an influx of calcium ions into vesicles of rod outer segments washed free of water-soluble proteins and filled with a high-affinity ligand for calcium (EDTA, Arsenazo III. aequorin). Addition of 3',5'GMP activates calcium influx without any appreciable delay studied with methods that resolve 40 ms. A direct interaction between the cyclic nucleotide and an intrinsic membrane component is considered. [ABSTRACT FROM AUTHOR]

The A and A* proteins of the bacteriophage 04 have been purified. The proteins have been analyzed for their enzymatic activities on single-stranded and double-stranded DNA. The A protein introduces a single-stranded break at a specific place in the G4 replicative form I DNA. This cleavage site has been localized between nucleotides 506 and 507 in the viral (+) strand. The A protein binds covalently to the 5' end of the cleavage site. The A protein initiates the replication of the viral (+) DNA [Borrias, et al. (1979) Virology, 31, 288-298]: the cleavage site therefore identifies the origin of replication. The A* protein cleaves viral (+) strand DNA at many different sites and also binds covalently to the 5' ends of the nick sites. The properties of both proteins strongly resemble the properties of the A and A* proteins of the related and much butter analyzed phage ∅X 174. These results indicate that the G4 and ∅XI 74 A and A* proteins have comparable functions and also that both phages initiate the replication of their replicative form DNA in a similar way. [ABSTRACT FROM AUTHOR]

Duck erythroblast nuclei, isolated under conditions designed to preserve lysosome integrity, were extracted to yield a bulk population of nuclear ribonucleoprotein (nRNP) particles which. when fractionated on sucrose gradients, was resolved into two classes of RNA-protein complexes containing low-molecular-weight RNA. The classical pre-messenger ribonucleoprotein complex (pre-mRNP), which had been previously characterized in this laboratory and was shown to contain rapidly-labeled pre-messenger RNA (pre-mRNA) sequences, was observed to contain approximately eight discrete species of nuclear RNA between 90 and 230 nucleotides in length (major species of Mr 80000 and 55000, and minor species of Mr 61000, 58000, 51000, 38000, 36000 and 32000). The size and relative distribution of these RNA sequences indicated that they were the small nuclear RNA species previously described in the literature. In addition, two smaller ribonucleoprotein complexes with sedimentation coefficients of 17 S and 12 S were characterized and shown to be composed of characteristic, although not mutually exclusive, sets of low-Mr RNA species. Comparison of these RNA species in the two smaller nRNP complexes with those found in the 40-S pre-mRNP particles demonstrated that all but three of the major low-Mr RNA bands found in the small nRNP were found in the pre-mRNP. The additional RNA species unique to the small nRNP were major ones of Mr 150000 and 110000, minor ones of Mr 48000, 34000 and 27000 and three major bands of Mr approximately 25000 as well. Further comparison revealed that while each particle was composed of a characteristic subset of polypeptides, several common proteins were observed in both small nRNP and pre-mRNP. The common RNA and polypeptide components observed in these two classes of nuclear ribonucleoprotein suggested that these small nRNPs may represent either integral parts of intact physiological pre-mRNP or nuclear structures which are closely associated with pre-mRNP in vivo. No difference was observed in either the RNA or protein composition of small nRNP or pre-mRNP when prepared in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. indicating that endogenous protease activity was not a problem in the preparation of nRNP from duck erythroblasts. CsCl gradient analysis of the two fractionated small nRNPs revealed buoyant densities of 1.39 and 1.40 g/ml for the IT-S and 12-S particles respectively. From these densities, an approximate RNA:protein ratio of 1:4, similar to that previously determined for the 40-S pre-mRNP complex, was estimated. Isoelectric focusing of the small nRNP on a pH gradient between pH 4 and pH 6 and electron microscopy studios reaffirmed the particulate nature of these smaller ribonucleoprotein complexes. In addition, ultrastructural analysis of small nRNP particles indicated an average diameter of 15 nm, compared with an average diameter of 35–40 nm for the bulk of the 40-S pre-mRNP particles. The poly(A)-protein complex of pre-RNA, sedimenting at a position of 15 S on the sucrose gradients, was bound to oligo(dT)-cellulose and subsequently eluted from the column in buffer containing 50% formamide. Six major proteins were associated with this poly(A) sequence, including the 73000-Mr poly(A)-binding protein typically seen associated with the poly(A) sequence of mRNA and pre-mRNA. In contrast, neither the 17-S nor the 12-S nRNP was bound to the oligo(dT)cellulose column indicating that these nRNPs did not contain poly(A) sequences and were, therefore, nRNP complexes distinctly different from the previously described poly(A)-protein complex of pre-mRNA. [ABSTRACT FROM AUTHOR]

Acetylcholine receptor-rich membrane fragments purified from Torpedo marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40000-Mr chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent molecular weights 43000, 50000 and 66000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these three peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was, in addition, stimulated up to 3-5-fold by K+. The effect of Na+ ions appeared less specific since Na+ ions reduced the labelling of all the poly- peptides susceptible to phosphorylation. Cholinergic agonists and antagonists, local anesthetics and cyclic nucleotides did not affect the phosphorylation of the receptor-rich membranes. Phosphorylation selectively modified the solubilization of several polypeptides by nondenaturing detergents: phosphorylated 43000-Mr, 50000-Mr and 66000-Mr polypeptides were solubilized at lower concentrations of detergent than their non-phosphorylated counterparts. Two-dimensional gels revealed the existence of a charge heterogeneity of the 40000-Mr and 43000-Mr chains. The microheterogeneity of the 43000-Mr chain, but not that of the 40000-Mr chain, might result from a selective phosphorylation of this particular chain. [ABSTRACT FROM AUTHOR]

Two groups of crosslinked polyacrylic gels with immobilized lysine and lysine peptides (Lys)5 and (Lys)5-Pro have been used as models for the chromatographic investigation of lysine-peptide-oligonucleotide interactions. One group carries carboxylic groups in addition to the peptide residues in the gel matrix; the other gel type contains no such carboxylic groups in the gel matrix. Nucleotides of the series (dT)2–5, p(dT)1–4, p(dT)1–4p, (dA)2–5, p(dA)1–5 and p(dA)1–4p were chromatographed on these gels under various conditions in an aqueous buffer. On the gels of the first group the nucleotides were retarded only slighly, the positive charges of the e-amino groups being compensated partially or totally by the negatively charged carboxyl groups of the polymer matrix. On the gels of the second group, however, the oligonucleotides underwent specific interactions. These interactions were based primarily upon electrostatic forces between the positively charged ϵ-amino groups of the immobilized peptides and the negatively charged phosphate groups of the oligonucleotides. Our results indicate that, in addition to the electrostatic interaction, the conformation plays a crucial role. We explain the selectivity of the interaction with a conformation-fit mechanism. The origin of this mechanism, which creates specific interactions from unspecific forces, is discussed. [ABSTRACT FROM AUTHOR]

Dormant and developing embryos of Artemia salina contain equivalent amounts of eIF-2, the eukaryotic initiation factor which forms a ternary complex with GTP and Met-tRNAf. The factor was purified from 0.5 M NH4Cl ribosomal washes by (NH4)2SO4 fractionation, followed by chromatography on heparin-Sepharose, DEAE-cellulose, hydroxyapatite and phosphocellulose. Purified preparations from dormant and developing embryos have similar specific activities and nucleotide requirements. The mobility of both proteins in dodecylsulfate gel electrophoresis is indistinguishable, and each contains three major polypeptide chains of molecular weight 52000, 45000 and 42000. Both proteins are also immunologically identical, and each stimulates amino acid incorporation in a cell-free system of protein synthesis. The binding of [35S]Met-tRNAf to 40-S ribosomal subunits is catalyzed by eIF-2 isolated from dormant or developing embryos and is dependent upon GTP and AUG. Binding of [35S]Met-tRNAf to 40-S ribosomal subunits, and ternary complex formation with eIF-2, GTP, and [35S]Met-tRNAf is stimulated 2–3-fold by a factor present in the 0.5 M NH4Cl ribosomal wash and which elutes from DEAE-cellulose at 50 mM KCl. This protein does not exhibit GTP-dependent binding of [35S]Met-tRNAf. Binding of GDP and GTP was investigated with purified eIF-2 from developing embryos. The factor forms a binary complex with GDP or GTP, and eIF-2-bound [3H]GDP exchanges very slowly with free nucleotides. Our results suggest that eIF-2 does not limit resumption of embryo development following encystment, nor does it limit mRNA translation in extracts from dormant embryos. [ABSTRACT FROM AUTHOR]

Incorporation of N-acetylglucosamine into endogenous lipid and protein acceptors was invest i- gated on heavy microsomes from rat liver, incubated with UDP-N-acetyl[14C]glucosamine and GOP-mannose in the absence of detergent. This subcellular preparation derived for 95 % or more from the rough endoplasmic reticulum and was devoid of Golgi components which contain the enzyme that adds the peripheral N-acetylglucosamine units to glycoproteins. The label was found almost exclusively in dolichyl diphosphate N-acetylglucosamine, except when the subcellular preparation was treated with pyrophosphate and subsequently incubated with the nucleotide sugars in the presence of GTP. Then, the incorporation of N-acetylglucosamine was considerably enhanced, and the additional label was associated with dolichyl diphosphate N,N'-diacetylchitobiose, with dolichyl diphosphate oilgosaccharides and with proteins The time-course of N-acetylglucosamine incorporation in these products was compatible with the pathway of dolichyl diphosphate glycoconjugates for the biosynthesis of the core portion of saccharide chains linked to asparagine residues of glycoproteins. The addition of GDP-mannose to the incubation medium was required to produce labeled dolichyl diphosphate oligosaccharides, but not to incorporate N-acetyl- glucosamine in protein. It is concluded that rough microsomes are capable of assembling dolichol-linked oligosaccharides from exogenous nucleotide precursors and of transferring N,N'-diacetylchitobiose, or its mannosylated derivatives, from the lipid intermediate to endogenous proteins. However, these metabolic activities are hindered in the original subcellular preparation, and in the absence of GTP. Although the earliest perceptible effect produced jointly by the treatment with pyrophosphate and by GTP was the synthesis of dolichyl diphosphate N,N'-diacetylchitobiose, the primary action of these factors remains uncertain. They may stimulate directly the reaction forming dolichyl diphosphate N,N'- diacetylchitobiose from dolichyl diphosphate N-acetylglucosamine, or activate the synthesis of this latter intermediate from a particular pool of dolichyl monophosphate which is readily converted afterwards into disaccharide and oligosaceharide derivatives and glycosylates protein. The requirement for GTP might have a functional meaning, for GTP acted maximally at a concentration distinctly lower than its actual concentration in liver. The detachment of ribosomes from rough vesicles was the major alteration induced by treatment with pyrophosphate. It is suggested that the removal of ribosomes unmasks the membrane sites where GTP acts. [ABSTRACT FROM AUTHOR]

Exchangeability of actin-bound ADP and calcium in actomyosin at low ionic strength has been studied using F-actin labelled with [14C]ADP or 45Ca and measuring release of radioactivity into solution. Low-speed centrifugation, ultracentrifugation and ultrafiltration were used to separate protein from the medium. Comparison of the results obtained with these three separation procedures has revealed that the release of [14C]ADP and 45Ca into the medium in the presence of millimolar concentrations of MgATP is largely due to the release under these conditions of actin itself retaining its bound ADP and calcium. The real exchange of the bound nucleotide and calcium, even under the most favourable conditions, was in our experiments limited to about 20 %. Detailed examination of the dependence of both the release of actin and the exchange of actin-bound ADP and calcium on the free divalent cations present, the kind and concentration of the added nucleotide, and temperature of incubation indicates that there is no correlation between the exchange and superprecipitation of actomyosin. The results presented support the view that the limited enhancement by myosin of the exchange of nucleotide and cation bound to actin under certain conditions results from accidental disruption of bonds between actin monomers due to a mechanical stress exerted on actin filaments upon their interaction with myosin filaments. [ABSTRACT FROM AUTHOR]

Limited digestion of the Escherichia coli 50-S subunit permits the isolation of two large fragments of the 23-S RNA. One arises from the 5' end of the 23-S RNA and contains about 1200 nucleotides. The other arises from the 3' end of the 23-S RNA and contains about 2000 nucleotides. Each of the ten 50-S proteins known to bind specifically and independently to the 23-S RNA were tested for their ability to interact with these two RNA fragments. It was determined that the 5' terminal segment contains the specific binding sites for proteins L4, L20 and L24, whereas the 3' terminal segment contains the specific binding sites for proteins LI, L2, L3, L6, L13, L16 and L23. [ABSTRACT FROM AUTHOR]

The nucleotide specificity of membrane-bound Na+- and K+-activated ATPase from ox brain was compared with that of the Na+-dependent phosphorylation reaction of ATPase protein. 32P]Phospbate is incorporated into enzyme protein from terminally-labelled [32P]nucleotide triphosphates (ATP, ITP, GTP), a process dependent on the presence of Na+. Subsequent addition of K+ ions to the test system is followed by a rapid decrease in incorporated radioactivity. Enzyme-bound phosphate exchanges its label rapidly with adenine nucleotides and slowly with inosine and guanosine nucleotides. ATP, ITP, and GTP are substrates of Na+- and K+-activated ATPase, The ratio of the reaction rates is ATP: ITP: GTP = 27: 2: 1 . An almost identical activation pattern of nucleotide triphosphate hydrolysis by Na+ ions in the presence of saturating concentrations of K+ is found with all substrates (Km = 10 mM NaCl). On the other hand, the optimal stimulation by K+ at saturating concentrations of Na+ is variable hydrolysis of ATP is increased 10 times, that of ITP 4 times and that of GTP 1.2 times, ADP inhibits both ATP and ITP hydrolysis. Only high concentrations of IDP (50 mM) inhibit ATP hydrolysis, whereas low concentrations of IDP inhibit ITP hydrolysis. Identical nucleotide triphosphate specificities were found for Na+ and K+activated ATPase from ox brain and from human red blood cells. It is concluded that all nucleotide triphosphates investigated are hydrolyzed via the same acceptor group. The Na+-dependent phosphorylation reaction of ATPase protein is considered as part of the overall reaction of membrane-bound Na+- and K+-activated ATPase. [ABSTRACT FROM AUTHOR]

Values for binding of ATP and ADP to myosin B and ATP to myosin A were determined from mixtures of these nucleotides and proteins by use of rapid filtration to separate bound and unbound nucleotides and by use of an ATP-regenerating system (ereatine phosphate­creatine phosphokinase). Calculation of the binding curves of ATP to 105 g of myosin B by the mass law binding expression using two sets of binding sites, n1 and n2, and with binding constants, k1=40 × 103 and k2=1.5 × 103 1/mole, respectively, yielded a curve of good fit to the experimentally determined points when n1=0.4 and n2=1.2 moles per l05 g. Similar calculation of data from binding of ATP to myosin A fielded a curve of best fit when n1=0.5 and n2=3.0 per 105 g with k1=5,500 and k2=550 1/mole. At 1 mM ATP, myosin A bound 50% more than myosin B. ADP at 1 mM appeared to saturate myosin B about 97%. The amounts of ADP bound to myosin B fitted a curve with one value of n which equaled 0.36. Multiplication of 105 g by the factors required to convert the above n values to integers fields combining weights of 250,000 and 200,000 for myosin B and myosin A. [ABSTRACT FROM AUTHOR]