bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of a truncated tobacco NtCBP4 channel in transgenic plants and disruption of the homologous Arabidopsis CNGC1 gene confer Pb2+ tolerance
Authors: Sunkar Ramanjulu , Kaplan Boaz , Bouché Nicolas , Arazi Tzahi , Dolev Dvora , Talke Ina N. , Maathuis Frans J.M. , Sanders Dale , Bouchez David , Fromm Hillel
Published: August 2010

Recently we reported on a plasma membrane tobacco protein (designated NtCBP4) that binds calmodulin. When overexpressed in transgenic plants, NtCBP4 confers Pb2+ hypersensitivity associated with enhanced accumulation of this toxic metal. To further investigate possible modulation of Pb2+ tolerance in plants, we prepared transgenic plants that express a truncated version of this protein (designated NtCBP4ΔC) from which its C‐terminal, with the calmodulin‐binding domain and part of the putative cyclic nucleotide‐binding domain, was removed. In contrast to the phenotype of transgenic plants expressing the full‐length gene, transgenic plants expressing the truncated gene showed improved tolerance to Pb2+, in addition to attenuated accumulation of this metal. Furthermore, disruption by T‐DNA insertion mutagenesis of the Arabidopsis CNGC1 gene, which encodes a homologous protein, also conferred Pb2+ tolerance. We suggest that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells.
The maize LAG1‐O mutant suggests that reproductive cell lineages show unique gene expression patterns early in vegetative development
Authors: Giedt Christopher D. , Weil Clifford F.
Published: August 2010

Patterns of transposable element activity often provide useful information about how and when organisms regulate gene expression. The maize lowered Ac/Ds germinal reversion 1 (LAG1)‐O mutation causes unusually low rates of germinal reversion by Ac/Ds‐induced alleles even though these same alleles revert frequently and early in somatic development. LAG1‐O suppresses Ds transposition at multiple, unlinked loci, and does not affect Spm elements, indicating that the mutation acts in trans and may be specific to Ac/Ds elements. Our data suggest that LAG1‐O suppression gradually reduces Ac/Ds activity in the meristem and newly formed leaves until, by the floral transition, transposition is undetectable even with PCR‐based assays. This suppression persists during tassel development and does not appear to be released until some point after meiosis. Competitive RT–PCR results show no difference in Ac transposase mRNA levels between LAG1‐O and lag1+ tassels, suggesting that suppression is post‐transcriptional. The pattern of LAG1‐O expression is consistent with a model in which at least some gene expression specific to those meristem cells that will ultimately give rise to floral tissue and therefore gametes begins very early in plant development, and then persists throughout development.
Identification of genes encoding receptor‐like protein kinases as possible targets of pathogen‐ and salicylic acid‐induced WRKY DNA‐binding proteins in Arabidopsis
Authors: Du Liqun , Chen Zhixiang
Published: August 2010

To understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen‐ and salicylic acid (SA)‐induced DNA‐binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W‐box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor‐like protein kinases (RLK), each consisting of an N‐terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C‐terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W‐box elements in its promoter region were recognized by both purified WRKY proteins and SA‐induced W‐box binding activities from SA‐treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein‐binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen‐ and SA‐induced W‐box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti‐microbial activities, but also genes encoding proteins with regulatory functions.
Carboxyl‐methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein
Authors: Rodríguez‐Concepción Manuel , Toledo‐Ortiz Gabriela , Yalovsky Shaul , Caldelari Daniela , Gruissem Wilhelm
Published: August 2010

Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post‐prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP‐labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl‐methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl‐cysteine methyltransferase (PCM) activity that carboxyl‐methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.
Monosaccharide/proton symporter AtSTP1 plays a major role in uptake and response of Arabidopsis seeds and seedlings to sugars
Authors: Sherson Sarah M. , Hemmann Georg , Wallace Graham , Forbes Susan , Germain Véronique , Stadler Ruth , Bechtold Nicole , Sauer Norbert , Smith Steven M.
Published: August 2010

The aim of this study was to investigate the in vivo properties and function of the high‐affinity monosaccharide/proton symporter AtSTP1 of Arabidopsis. We isolated an Atstp1 knock‐out mutant and found that this plant grows and develops normally. The AtSTP1 gene is expressed in germinating seeds and seedlings, with AtSTP1 activity found mainly in the seedling root. The rate of uptake of [14C]‐3‐O‐methylglucose and [14C]‐d‐glucose is 60% less in Atstp1 seedlings than in the wild type, showing that AtSTP1 is the major monosaccharide transporter in Arabidopsis seedlings. Transport of d‐galactose and d‐mannose is also up to 60% less in Atstp1 seedlings compared to wild type, but transport of d‐fructose, l‐arabinose and sucrose is not reduced. Germination of Atstp1 seed shows reduced sensitivity to d‐mannose, demonstrating that AtSTP1 is active before germination. Atstp1 seedlings grow effectively on concentrations of d‐galactose that inhibit wild‐type growth, even at up to 100 mm d‐galactose, indicating that active transport by AtSTP1 plays a major role at very high concentrations of exogenous sugar. These findings provide insight into the physiological function of AtSTP1 and clearly establish its importance in the uptake of extracellular sugars by the embryo and in seedlings.
Double‐stranded RNA interferes with gene function at the single‐cell level in cereals
Authors: Schweizer Patrick , Pokorny Jana , Schulze‐Lefert Paul , Dudler Robert
Published: August 2010

Double‐stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence‐specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co‐bombarded UidA (GUS) and TaGLP2a::GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol‐4‐reductase in maize and barley, respectively. Dihydroflavonol‐4‐reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1‐ and b‐Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol‐4‐reductase gene, C1‐ and b‐Peru‐dependent, cell‐autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non‐specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss‐of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence‐specific interference with gene function at the single‐cell level.
Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity
Authors: Gallego Maria E. , Jeanneau M. , Granier Fabienne , Bouchez David , Bechtold Nichole ,   Charles, White I.
Published: August 2010

The Rad50 protein is involved in the cellular response to DNA‐double strand breaks (DSBs), including the detection of damage, activation of cell‐cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino‐ and carboxy‐terminal regions. Mutant plants present a sterility phenotype which co‐segregates with the T‐DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T‐DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA‐damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double‐strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50–Mre11–Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.
Nod factor‐induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation
Authors: Den Hartog Martine , Musgrave Alan , Munnik Teun
Published: August 2010

Rhizobium‐secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [32P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.
The jasmonate‐inducible AP2/ERF‐domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate‐responsive promoter element
Authors: Van Der Fits Leslie , Memelink Johan
Published: August 2010

The AP2/ERF‐domain transcription factor ORCA3 is a master regulator of primary and secondary metabolism in Catharanthus roseus (periwinkle). Here we demonstrate that ORCA3 specifically binds to and activates gene expression via a previously characterized jasmonate‐ and elicitor‐responsive element (JERE) in the promoter of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str). Functional characterization of different domains in the ORCA3 protein in yeast and plant cells revealed the presence of an N‐terminal acidic activation domain and a serine‐rich C‐terminal domain with a negative regulatory function. Orca3 mRNA accumulation was rapidly induced by the plant stress hormone methyljasmonate with biphasic kinetics. A precursor and an intermediate of the jasmonate biosynthetic pathway also induced Orca3 gene expression, further substantiating the role for ORCA3 in jasmonate signaling. The protein synthesis inhibitor cycloheximide did not inhibit jasmonate‐responsive expression of Orca3, nor of its target genes Str and Tryptophan decarboxylase (Tdc). In conclusion, ORCA3 regulates jasmonate‐responsive expression of the Str gene via direct interaction with the JERE. The activating activities of ORCA proteins do not seem to depend on jasmonate‐induced de novo protein synthesis, but presumably occur via modification of pre‐existing ORCA protein.
Spatial and temporal expression of two cytosolic glutamine synthetase genes in Scots pine: functional implications on nitrogen metabolism during early stages of conifer development
Authors: Avila Concepción , Suárez María Fernanda , Gómez‐Maldonado Josefa , Cánovas Francisco M.
Published: August 2010

Ammonium assimilation during the initial stages of Scots pine growth involves two cytosolic glutamine synthetase (GS, EC: 6.3.1.2) isoenzymes encoded by separate genes, GS1a and GS1b. GS1a was most exclusively expressed in photosynthetic tissues of the seedling whereas GS1b was expressed ubiquitously showing higher levels in non‐photosynthetic tissues such as root and hypocotyl. Temporal expression analysis has shown that when germination starts GS1b is the predominant form in the embryo, however, its relative abundance in the tissue decreased in the postgerminative stages when green cotyledons are developed. In contrast GS1a was present at a low level in the embryo but its abundance increased markedly during germination and seedling growth. These data suggest that GS1a and GS1b genes display different and non‐redundant roles in the nitrogen metabolism of conifers. The precise localization of individual transcripts by in situ hybridization strongly supports this possibility. GS1 gene products are mainly expressed in different cellular types: GS1a in chlorophylic parenchyma and GS1b in the vascular bundles of all tissues examined in the seedling. Our data support that glutamine biosynthesis in pine seedlings follows a different pattern related to angiosperms involving two cytosolic GS proteins: one of them a typical cytosolic GS which may be involved in the generation of glutamine for N transport and a second cytosolic GS generating amino donors for the biosynthesis of major N compounds in photosynthetic tissues, a closer role to angiosperm chloroplastic GS. The results are discussed with regard to recent studies on N mobilization and metabolism during the initial stages of conifer development.
Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase
Authors: Mauch Felix , Mauch‐Mani Brigitte , Gaille Catherine , Kull Beatriz , Haas Dieter , Reimmann Cornelia
Published: August 2010

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease‐resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate‐lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB‐A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c‐SAS plants) or to the chloroplast (p‐SAS plants). In p‐SAS plants, the amount of free and conjugated SA was increased more than 20‐fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P‐SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR‐1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c‐SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.
Interaction of pollen‐specific actin‐depolymerizing factor with actin
Authors: Smertenko Andrei P. , Allwood Ellen G. , Khan Safina , Jiang Chang‐Jie , Maciver Sutherland K. , Weeds Alan G. , Hussey Patrick J.
Published: August 2010

We have examined the interaction of recombinant lily pollen ADF, LlADF1, with actin and found that whilst it bound both G‐ and F‐actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil – a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F‐actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF : actin array is replaced by actin : ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co‐localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.
ISOLATION OF NON‐IDENTICAL POLYPEPTIDE CHAINS OF α‐CRYSTALLIN
Authors: Schoenmakers J. G. G. , Matze R. , Poppel M. van , Bloemendal H.
Published: August 2010

α‐Crystallin–a water‐soluble lens protein–was dissociated into subunits by means of urea treatment at pH 3. The dissociated protein was resolved into three distinct polypeptide species by chromatography on SE‐Sephadex columns at pH 3.2 equilibrated with 7 M urea.

In the presence of urea the isolated polypeptides, designated as I‐a, I‐b, and II, have different electrophoretic mobilities at acid and alkaline pH. It could be demonstrated that two cysteine residues are present in polypeptide I‐a whereas cysteine is absent in polypeptide II. Moreover, amino‐acid analysis reveals that both polypeptides have a different amino‐acid composition. The third polypeptide has shown to be a dimer of one of the polypeptides. This dimer arises during isolation by disulphide bond formation. The results indicate that a‐crystallin is composed of two distinctly different kinds of subunits. Evidence is presented that both types are built up by two different polypeptide chains.
STUDIES ON THE DENATURATION OF TROPOMYOSIN AND LIGHT MEROMYOSIN
Authors: Woods E. F.
Published: August 2010

Optical rotatory dispersion has been used to study the heat denaturation of tropomyosin and light meromyosin fraction 1 (LMMFr I) as a function of pH and salt concentration. The unfolding of these highly a‐helical molecules takes place in stages indicating regions of varying stability along the molecule. At all pH values NaCl and KCl increase the stability and this is attributed to electrostatic screening of charged groups. There is a decrease in stability to heat at alkaline pH values and the molecules are most stable on the acid side of the iso‐electric point.

Guanidine hydrochloride is more effective than urea as a denaturant. In the case of tropomyosin almost complete reversibility of the optical rotation changes by these denaturants can be achieved provided the ‐SH groups are prevented from forming disulphide bonds. For LMMFr I the rotation changes and reversibility are complicated by depolymerization into smaller units. Molecular weight determinations by sedimentation equilibrium indicate a critical urea concentration for depolymerization which is not evident from the optical rotation changes. Although guanidine hydrochloride is more effective than urea in abolishing the helical structure of LMM Fr 1, it is not as effective as a depolymerizing agent.
CHROMATOGRAPHIC STUDIES ON HUMAN RED CELL PHOSPHOGLUCOMUTASE
Authors: Monn Eirik
Published: August 2010

Human red cell phosphoglucomutase of the three common phenotypes have been examined by gel filtration and ion‐exchange chromatography. By gel filtration on Sephadex G‐100 the molecular weight of all the PGM1 components was estimated to be about 51,000 and all the PGM2 components to be about 58,000. Experiments on DEAE cellulose indicate that the different phosphoglucomutase components differ in charge. Probably, the components are present in the haemolysate and not produced as artefacts during separation procedures. The possible biochemical basis for the differences among the components related to each allele is discussed.
THE INTRODUCTION OF SYNTHETIC CROSS‐LINKS INTO TROPOCOLLAGEN AND THEIR EFFECTS ON THE THERMAL STABILITY OF THE MOLECULE
Authors: Woodlock Anne F. , Harrap B. S.
Published: August 2010

Tropocollagen from rat‐tail tendon has been reacted with 33 per cent basic chromic sulphate (empirical formula CrOHSO4) to give soluble reaction products. Thermal denaturation of the products and examination in the ultracentrifuge has shown the appearance of ‐γ‐components suggesting that chrome cross‐links have been introduced into the collagen molecule. Further evidence for cross‐linking has been obtained from studies of renaturation of the thermally denatured reaction products.

A study has been made of the effects of both temperature and chrome I collagen ratios on the thermal stability of the reaction products, characterized by their thermal transition temperatures, Tm, and on their contents of irreversibly bound chromium. The value of Tm decreased as the reaction proceeded in the initial stages but in most cases passed through a minimum to increase in the later stages. The amount of irreversibly bound chromium increased continuously to a plateau with time of reaction. These data are interpreted in terms of (A) the increase in net charge on the protein as the cationic chrome complex reacts with protein carboxylate groups and (B) the introduction of cross‐links into a previously ordered conformation (Flory 1956).
HETEROGENEITY OE DEFATTED BOVINE SERUM ALBUMIN
Authors: Andersson Lars‐Olov
Published: August 2010

The heterogeneity of bovine serum albumin has been studied with respect to solubility in 3.0 M KCl in the pH‐range 4.7‐3.9. Both the ‐SH heterogeneity and adsorbed contaminants affect the pH‐solubility curves. Mercaptalbumin defatted by charcoal treatment was found to be completely homogeneous with regard to pH‐solubility properties.
ON THE CONFORMATION OF BOVINE SERUM ALBUMIN AFTER ALKALINE OR THERMAL DENATURATION
Authors: Harmsen B. J. M. , Braam W. G. M.
Published: August 2010

An α‐helix to β‐form transition has been suggested in bovine serum albumin upon irreversible denaturation by alkali or heat. Since the optical properties of the β‐con‐formation are better known at present a renewed study was made of the structure of bovine serum albumin after alkaline or thermal denaturation by means of optical rotatory dispersion and infrared absorption measurements. Electrophoresis on poly‐acrylamide gels and gelfihration on Sephadex G‐100 or G‐I50 were employed to investigate the extent of aggregation ami the kind of binding which stabilizes the aggregates formed after denaturation by heat. The results indicate that the α‐helical conformation of bovine serum albumin is rather resistant to denaturation. Only a partial transition from α‐helix to random coil was found without the introduction of a new conformation. This transition seemed to be at least partially reversible upon cooling after thermal denaturation or upon neutralization after alkaline denaturation.
PREPARATION OF SOME NEW BIPHENYLISOPROPYLOXYCARBONYL AMINO ACIDS AND THEIR APPLICATION TO THE SOLID PHASE SYNTHESIS OF A TRYPTOPHAN‐CONTAINING HEPTAPEPTIDE OF BOVINE PARATHYROID HORMONE
Authors: Wang Su‐Sun , Merrifield R. B.
Published: August 2010

Several new crystalline 2‐p‐biphenyl‐2‐propyloxycarbonyl (Bpoc) amino acids were synthesized and characterized. They were found to he stable for months as free acids if kept cold and dry. The Bpoc group could be quantitatively removed by anhydrous 0.5% trifluoroacetic acid in methylene chloride within 10 minutes, under conditions where side chain N‐benzyloxycarbonyl groups, O‐benzyl ethers, or benzyl esters were completely stable. The tryptophan‐containing peptide, H‐Glu‐Arg‐Val‐Glu‐Trp‐Leu‐Arg‐OH, of bovine parathyroid hormone, was synthesized in good yield by the automated solid phase method using the Bpoc‐amino acids.
THE EFFECT OF ALIPHATIC ALCOHOLS ON THE THERMAL STABILITY OF TROPOCOLLAGEN UNDER ACIDIC CONDITIONS
Authors: Harrap B. S.
Published: August 2010

Monohydric and dihydric alcohols have been added to aqueous solutions of rat tail tendon collagen at pH 3.0 and their effects on the thermal denaturation temperature, Tm, of the protein have been studied. At concentrations less than 2 M all the monohydric alcohols gave linear curves of Tm vs alcohol concentration with negative slopes. The extent of lowering of Tm increased with increasing Cn for the n‐alcohols from C1, to C4. An alcohol with its carbon chain branched at the α‐carbon atom lowered Tm less than its straight‐chain isomer but branching at the β‐carbon atom produced little effect. At high alcohol concentrations positive deviations from linearity were observed in the Tm vs c curves indicating a competitive stabilizing effect. Of the dihydric alcohols, ethylene glycol and propane‐1:3‐diol each increased Tm at all concentrations whereas propane‐J: 2‐diol decreased Tm except at very high concentration (< 10 M).

The addition of ethanol (20% v/v) had only a small effect on the relations between both Tm and pH and Tm and concentration of added salts at pH 3.0.

It is concluded from these data that addition of alcohols has only small effects on the contribution to the stability of tropocollagen from electrostatic interactions but major effects on the contributions from hydrogen‐ and hydrophobic‐bonding. The net effect appears to depend primarily on the hydrophobic‐hydrophilic balance and steric factors in the alcohol molecule.
CORRELATIONS BETWEEN AMINO ACID SEQUENCE AND CONFORMATION OF IMMUNOGLOBULIN LIGHT CHAINS
Authors: Welscher H. D.
Published: August 2010

A detailed comparison of the primary structure of 15 individual immunoglobulin light chains including 6 human k chains, 2 mouse k chains, and 7 human. chains is given. Their chemical similarities and the possible conformational influence of the invariant residues are discussed. Some preliminary results on heavy chains are also considered. The most striking structural feature common to all L chains examined is the pattern of distribution of nonpolar, but not of polar or ionized residues along the sequences. Both variable (VL) and constant (CL) regions of L chains contain approximately 55 nonpolar residues. Out of these, 30 nonpolar residues are found in all VL, regions at structurally identical sequence sites and 33 in all CL regions. This frequency of consistently nonpolar residues is similar to that in globin chains and is probably due to thermodynamical and stereochemical requirements. The globular folding of L chains is essentially stabilized by hydrophobic interactions and most of the consistently nonpolar sequence positions are likely to represent intrachain hydrophobic contact sites. These, however, require well‐defined distances between the interacting groups. There is no significant similarity between the pattern of nonpolar residues of the VL, and Ch regions. However, the rule of isopolar substitution suggests a similar folding of all VL, (and possibly Vrd regions, on the one hand, and a similar folding of all CL (and possibly CH) regions, on the other. The low a‐helical content of L chains is explained, and some conclusions concerning future structural studies are drawn.
AMINO ACID SEQUENCES OF ALL THE TRYPTIC PEPTIDES FROM THE α POLYPEPTIDE CHAIN OF ADULT HEMOGLOBIN OF THE RHESUS MONKEY (Macaca mulatto)
Authors: Matsuda Genji , Maita Tetsuo , Igawa Nagatoshi , Ota Hisahiro , Miyauchi Takao
Published: August 2010

The a polypeptide chain of adult hemoglobin of the rhesus monkey was digested with trypsin. All the tryptic peptides thus obtained were isolated and purified by column and paper chromatography and amino acid sequence analyses of these peptides were performed mainly by partial hydrolyses with enzymes, the DNP method, and the PTC method. The comparison of the results with the known sequences of the corresponding peptides from the a polypeptide chain of human hemoglobin showed that there were four differences in amino acid sequences of these peptides between rhesus monkey and humans.
ISOLATION and CHARACTERIZATION OF PEPTIDES PRODUCED BY THE MILD ACID HYDROLYSIS OF BOVINE SUBMAXILLARY MUCIN*
Authors: Downs Fred , Pigman Ward
Published: August 2010

The rate of release of amino acids during partial acid hydrolysis of bovine submaxillary mucin has been studied. A number of di‐ and tripeptides have been isolated on the amino acid analyzer. Three β‐hydroxyamino acid peptides, Thr‐Thr‐Thr, Ser‐Ser, and Thr‐Ser have been isolated and account for 12 % of these amino acids in native mucin. A substantial amount of the glycine and alanine has been isolated as dipeptides of serine and threonine with the fihydroxyamino acids in the amino‐terminal position. Proline, valine, isoleucine, and leucine seem to be in close association in the peptide core. The same pattern of peptides has been obtained from the partial acid hydrolysis of ovine submaxillary mucin.
AMINO ACID SEQUENCE IN ALL THE TRYPTIC PEPTIDES FROM THE β POLYPEPTIDE CHAIN OF ADULT HEMOGLOBIN OF THE RHESUS MONKEY (Macaca mulatta)
Authors: Matsuda Genji , Maita Tetsuo , Yamaguchi Masato , Migita Masaharu
Published: August 2010

The β polypeptide chain of adult hemoglobin prepared from the rhesus monkey (Macaca mulatta) was digested with trypsin. All the tryptic peptides thus obtained were isolated and purified by column chromatography and paper chromatography, and then the amino acid sequence in these peptides was determined. Comparisons of the results with the known sequences of the corresponding peptides from adult human hemoglobin showed there were differences at eight positions in the amino acid sequences of the seven peptides.
ON THE TRYPTOPHAN CONTENT OF NEOCARZINOSTATIN
Authors: Maeda Hiroshi , Meienhofer Johannes
Published: August 2010

Two tryptophan residues were determined in the polypeptide antitumor antibiotic neocarzinostatin using 2‐hydroxy‐5‐nitrobenzyl bromide (HNB) and 2‐nitrophenyl‐sulfenyl chloride (NPS‐Cl) in 50 % acetic acid.
STUDIES ON INHERITED ANTIGENIC VARIATION OF HUMAN SERUM β‐LIPOPROTEIN BY PASSIVE HEMAGGLUTINATION
Authors: Boman H.
Published: August 2010

A methodological study on passive hemagglutination and passive hemagglutination inhibition reactions with human serum β‐lipoprotein coupled to human red cells by bis‐diazotized benzidine has been performed. Several variable factors have been studied, and a standardized procedure for optimal, reproducible results is described. A diluent containing dextrose which was found superior to several other diluents examined is described.

Genetical differences in purified β‐lipoprotein were clearly demonstrated by the passive hemagglutination technique using human isoantisera, and routine typing can be performed by inhibition experiments. The passive hemagglutination and the hemagglutination inhibition tests described are specific and highly sensitive and are expected to become important supplements to the immunoprecipitation technique in the study of inherited β‐lipoprotein variants.

This work was supported by grants from J. L. Tiedemanns Tobaksfabrik, Joh. H. Andresens medisinske fond, C. H. Homans legat og legatet til Henrik Homans Minde, and Dahls legat to the author, as well as by grants from The Norwegian Research Council for Science and the Humanities, and from Nordisk Insulinfond, to Professor Kare Berg.
STUDIES ON BACTERIAL PROTEASES
Authors: Tsuru D. , Yoshida T. , Hirose T. , Yoshimoto T. , Fukumoto J.
Published: August 2010

The neutral subtilopeptidase amylosacchariticus, a zinc metalloprotease, obtained from Bacillus subtilis var. amylosacchariticus was treated with chemical reagents which were known to modify tyrosyl residues of proteins. Acetylation of about 14 of 23 tyrosine residues of the enzyme with acetylimidazole led to almost complete losses of activities toward casein and synthetic peptide, Z‐Gly‐L‐Leu. NH2, and the activities lost were fully restored by the additional treatment with hydroxylamine. lodination of about three tyrosine residues also caused almost complete loss in the caseinolytic activity but increased the activity toward Z‐Gly‐L‐Leu. NH2 threefold over that of the native enzyme. Quite similar results were obtained when about three tyrosine residues were nitrated with tetranitromethane. Kinetic analysis indicated that Km value for Z‐Gly‐L‐Leu. NH2 remained approximately unchanged with the native, acetylated, iodinated, and nitrated enzymes, while KCat(Kmax/E) varied markedly depending upon the type of modification and the number of tyrosyl residues modified, increasing with nitration and iodination but decreasing with acetylation of tyrosyl residues. These results suggest that some tyrosine residues together with a zinc atom are closely related to the enzyme action.
STUDIES ON THE CONFORMATION OF AMINO ACIDS
Authors: Ponnuswamy P. K. , Sasisekharan V.
Published: August 2010

Short range intramolecular interaction energies of a few amino acids with γ‐atoms are reported and their role in determining the molecular conformation has been again stressed. In serine and threonine, it is unlikely that the Oγ atom will occupy position II in which it is trans to the amino nitrogen. In cysteine, all three staggered positions of Sγ about the bond Cα‐Cβ become equally favourable. In valine, there is definite correlation between the orientation of the carboxyl group and that of the two γ‐methyl groups. In general, the backbone conformations of these amino acids are very similar to those of aminobutyric acid. The results indicate that even in amino acids containing hetero‐atoms in the side group where strong inter molecular interactions are possible, the molecular conformation is determined to a great extent by the intramolecular short range interactions alone.
HEMOGLOBIN ATAGO (α852 Tyr β2) A NEW ABNORMAL HUMAN HEMOGLOBIN FOUND IN NAGASAKI
Authors: Fujiwara Naoko , Maekawa Tomoyuki , Matsuda Genji
Published: August 2010

An abnormal human hemoglobin was discovered in a woman living at Atago in Nagasaki and was designated Hb Atago.

In the thin‐layer starch gel electrophoresis with Tris‐EDTA‐borate buffer, pH 8.6, the main component of this abnormal hemoglobin migrated towards the anode a little behind Hb F, and the minor abnormal component migrated to the same site as non‐hemoglobin protein did. These abnormal components were isolated by CM‐cellulose column chromatography. The hybridization method proved that the α chain of Hb Atago had the abnormality. The abnormal α chain was isolated by CM‐cellulose column chromatography in 8 M urea. The tryptic peptide map of the aminoethylated abnormal α chain showed that the abnormality existed in αT9. The abnormal αT9 was isolated and purified by high voltage paper electrophoresis. The abnormal chymotryptic peptides from αT9 were purified by high voltage paper electrophoresis and paper chromatography. The amino acid compositions of these peptides were analyzed.

As a result, it was found that in Hb Atago, a tyrosine residue was substituted for the aspartic acid residue at α85 in the normal human hemoglobin.
HYDROGEN ION TITRATION OF IONIZABLE SIDE‐CHAINS IN NATIVE AND DENATURED GLYCI.NIN
Authors: Catsimpoolas N. , Berg T. , Meyer E. W.
Published: August 2010

Electrometric and spectrophotometric titration curves of glycinin were obtained at 0.4 ionic strength and in the presence of 6M guanidine hydrochloride and 6M urea. The electrometric hydrogen ion titration curves were irreversible in the pH range 6.5 to 12.0 in 0.4M KCl, but exhibited reversibility in the pH 2.0 to pH 6.5 range. The forward and backward electrometric titration curves were entirely reversible in 6M guanidine hydrochloride. The spectrophotometric titration curves of tyrosine groups were also irreversible in 0.4M KCl, but reversible in 6M urea. Alkali‐induced denaturation produced a different spectrophotometric titration curve than the curves obtained with glycinin in 0.4M KCl and 6M urea. The electrostatic factor w was lower in the pH 2.50 to 3.75 range than in the pH 3.75 to 6.50 range. This was interpreted to be due to swelling and dissociation into subunits of the glycinin molecule subjected to acid denaturation. The empirical value of w=0.045 derived from the titration of the carboxyl groups in the pH 3.75 to 6.50 range coincided with the theoretically calculated value of 0.040 for a swollen sphere. Group counting and pK values of carboxyl, imidazole, e‐amino, and phenoxy groups under normal and denaturing conditions are also reported.
HUMAN PITUITARY GROWTH HORMONE. * XXIII.
Authors: Li Choh Hao , Chung David
Published: August 2010

Five protected peptide fragments occurring in the firsst 27 aniino acid rcsidiies of the HGH molecule have been spithesized miit characteriied. These peptides are:

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HUMAN PITUITARY GROWTH HORMONE.* XXIV.
Authors: Danho Waleed , Li Choh Hao
Published: August 2010

Four protected peptide fragments occurring in the regions of residues 28‐52 in the HGH molecule have been synthesized and characterized. These peptides are:

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HUMAN PITUITARY GROWTH HORMONE. * XXV
Authors: Kovacs Kalman , Kovacs‐Petres Yolande , Li Choh Hao
Published: August 2010

Three protected peptide fragments occurring in the region of residues 53‐67 in the HGH molecule have been synthesized and characterized. These peptides are

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HUMAN CERULOPLASMIN AS A POLYMORPHIC GLYCOPROTEIN
Authors: Rydén Lars
Published: August 2010

Commercial preparations of human ceruloplasmin were chromatographed on hydroxyapatite by the gradient elution technique. From two batches originating from different Cohn fractions, five forms of the protein were isolated. One of these appeared upon aging of one of the batches. Chemical analysis showed that the ceruloplasmin forms differed only in their carbohydrate content, with the exception of two of the forms (IIa and IIb) which had identical chemical composition. The total carbohydrate content of the ceruloplasmin forms varied between 6% and 10%. Two of the forms (IIa and IIb) were supposed to represent conformational variants of native ceruloplasmin components. It is concluded that at least three forms of ceruloplasmin are present in human serum. The polymorphism of human ceruloplasmin is discussed in the light of recent examinations of other glycoproteins.
THE REACTION OF CREATINE KINASE WITH DITHIOBISNITROBENZOIC ACID
Authors: O'Sullivan W. J.
Published: August 2010

Two derivatives of creatine kinase (CrK) have been formed by reaction with the sul‐phydryl group reagent, dithiobisnitrobenzoic acid (DTNB). In the first derivative, TNB(2) ‐CrK, the two essential ‐SH groups of the active dimer form were blocked. The inactivated enzyme had similar physico‐chemical properties to the native enzyme, including the ability to bind manganese‐nucleotide substrates. Full activity could be restored by treatment with dithiothreitol. It is suggested that TNB(2)‐CrK could serve as a convenient means of protecting the essential ‐SH groups of CrK, while the effects of other group‐specific reagents are explored.

A second derivative, TNB(8)‐CrK, was formed by carrying out the reaction in the presence of 8 M urea. Under these conditions, the CrK dimer is dissociated into monomers with exposure of a further six ‐SH groups. After removal of the urea, a significant concentration of the protein remained in solution in the monomer form. This derivative bound Mn2+ strongly but the nucleotide binding site was destroyed. A role for the “buried” sulphydryl groups in the formation of the stable dimer form of creatine kinase is indicated.
MOUSE INSULINS — SEPARATION AND STRUCTURES
Authors: Markussen Jan
Published: August 2010

Two major components were detected by disc electrophoresis in twice crystallized mouse insulin. Gel filtration experiments indicated that the two components were two insulins rather than proinsulin and insulin. The components were separated by ion‐exchange chromatography, and amino acid analysis showed both components to be insulin, differing in two amino acids. The mouse insulins were named Mouse I and Mouse II insulin by analogy with Rat I and Rat II insulin in accordance with the electrophoretic mobilities. Performic acid oxidation followed by separation of the A and B chains, and amino acid analysis of the separated chains showed both differences to be present in the B chains whereas the A chains were identical. Trypsinization of the B chains followed by separation of the tryptic peptides and amino acid analysis of the peptides yielded the following results: B 1 to B 3 were identical. B 4 to B 22 differed in one amino acid, mouse I having one proline and no serine, mouse II having one serine and no proline. Finally, free serine B 30 plus a heptapeptide (B 23‐B 29) containing one lysine and no methionine was obtained from mouse I, whereas mouse II yielded an octapeptide (B 23‐B 30) containing one methionine and no lysine.

Mouse II insulin displayed amino acid compositions identical with those of rat II insulin in regard to the A chain, the B chain, and the tryptic peptides from the B chain.
HOMOLOGY OF A PARTIAL SEQUENCE OF CALF THYMUS HISTONE IV WITH SEVERAL NON‐HISTONE PROTEINS
Authors: Bauer Klausdieter
Published: August 2010

A systematic analysis of the calf thymus histone IV sequence revealed the existence of two groups of internal homologies, A and B, comprising a total of 88 out of 102 amino acid residues. To test the hypothesis that present‐day proteins have developed from ancestral small‐size peptides by gene duplications and related mechanisms, we investigated a number of selected sequences from several non‐histone proteins for the occurrence of homologies with one of the homology groups mentioned. Comparisons between random sequences were employed as controls for this purpose. Homology with A was found in partial sequences of histone III and ferredoxin, probable homology in portions of cytochrome c, haptoglobulin β‐chain, chymotrypsinogen A, and two immunoglobulin H‐chains. This result may indicate common ancestry for all these molecules, dating back to an early period with small‐size peptides predominating. The implications of the findings for molecular evolution are discussed.
STRUCTURAL CHANGES INVOLVED IN THE FOLDING OF PROINSULIN
Authors: Markussen Jan
Published: August 2010

The circular dichroism spectra of reduced proinsulin (precursor of proinsulin), reduced insulin (A plus B chain), and C‐peptide, all of bovine origin, were recorded. The difference spectrum between reduced proinsulin and the sum of reduced insulin and C‐peptide was constructed. The difference spectrum indicates the presence of structures which may be those necessary to keep the proinsulin precursor in position for correct formation of the three cystine bridges of proinsulin and insulin. The difference spectrum can be interpreted as being due to approximately 6 additional amino acids in theα‐helix and 9 additional amino acids in theβ‐structure in reduced proinsulin, all 15 being in random coil state in the separated chains.
STUDY OF HYDROGEN BONDS IN AMINO ACIDS AND PEPTIDES*
Authors: Ramakrishnan C. , Prasad Nageshwar
Published: August 2010

An analysis of the various parameters associated with N‐H… O type of hydrogen bonds has been made using data from reported crystal structures of amino acids and simple peptides. The different parameters at the donor and the acceptor ends have been suitably defined and evaluated. In some cases the analysis is done depending upon the chargedness or other characteristics of the donor and acceptor groups. Histograms giving the distribution of these parameters have been drawn and possible conclusions arrived at:

1. The distribution shows a maximum between 2.8 A and 2.9 A for the charged donor group and 2.9 A and 3.0 A for the uncharged donor group and is probably not dependent upon the charge on the acceptor group.

2. The angle between the directions CO and O. N tends to lie between two cones about C O with semi‐vertical angles 40 and 70°. The orientation of the directions O. N and O. H with respect to the lone pair orbital directions on the acceptor oxygen atoms are analysed in detail using spherical polar coordinates. The analysis indicates that the group NH has a very strong tendency to point towards the acceptor oxygen atom. A general feature has been found in that the direction N‐H tends to be closer to an orbital if the oxygen is an acceptor of two hydrogen bonds, while the direction tends to lie in between the orbitals when the acceptor oxygen is the receipient of only one hydrogen bond. The possible explanation of this on the basis of lone pair interaction is briefly discussed.
PROTEIN CATABOLISM: ACTIVITIES OF THREE PROTEOLYTIC ENZYMES IN A SYNCHRONIZED L5178Y MOUSE LEUKEMIC CELL LINE
Authors: Bosmann H. Bruce
Published: August 2010

Exponential L5178Y cells had the following proteolytic activities (in μg pronase equivalents per hour per mg protein): general proteolytic (azocoll substrate), 120; cathepsin, 42; trypsin, 21; there was no detectable collagenase activity. The general proteolytic activity was distributed as a double peak enzyme with peaks in M and mid‐S. Cathepsin was distributed as a double peak enzyme with a narrow peak in mid‐S and high activity in M. Trypsin was distributed in a pattern which was described as either a double peak enzyme or as a continuous enzyme with depressions of activity occurring in early S and G2.