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The complete primary structure of the constant part of the μ-chain-disease protein, BOT, was established. It includes the whole CH2, CH3 and CH4 domains. Two amino acid changes were found, at positions 309 (Ser ↵ Gly) and 333 (Val ↵ Gly) (GAL numbering). In two additional monoclonal n chains (SCO and CO), the same positions showed an amino acid variability. From these data it may be concluded that four types of μ chains exist in the human: (1) GAL type with Ser-309 and Val-333; (2) OU type with Gly-309 and Val-333; (3) SCO type with Ser-309 and Gly-333; (4) BOT/CO type with Gly-309 and Gly-333. The meaning of this molecular polymorphism is discussed. [ABSTRACT FROM AUTHOR]

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]

1. Different types of bacteriorhodopsin chromophores and their reactions are described. This includes intermediates of the reconstitution reaction of the purple complex, intermediates of the photochemical cycle and a photochemically active 500-nm chromophore. In contrast to the native chromophore (purple complex) some of these species are reducible by borohydride yielding stable retinyl proteins. 2. Hydrolysis and/or extraction of the retinyl proteins reveals that in the corresponding non reduced parent chromophores the retinal is bound either noncovalently or covalently to lysyl residues of the polypeptide chain. 3. Retinol, retinyl lysine and their retro isomers isolated from the various reduced chromophores are identified by thin-layer chromatography and mass spectrometry. 4. The absorption spectra of the retinyl proteins and binding studies with retinol and retinal indicate that the cyclohexene ring and the side chain of the retinyl moiety are forced into a coplanar conformation by interaction with the protein. The three peaked absorption band of the reduced chromophores is due to this planarized conformation and not to a retro configuration of the retinyl moiety. 5. Isomerization to retro compounds can be achieved by HCl as is known to be the case for retinyl compounds in solution. In addition photochemical isomerization is observed in the case of planarized retinyl moieties. Thus the native protein structure is responsible not only for a specific conformation of the retinyl moiety but also for its specific reactivity. [ABSTRACT FROM AUTHOR]

The primary structure of the most acidic (pI = 4.50) of the two major parvalbumins from frog (Rana esculenta) has been determined from a study of its trypsic peptides and of overlapping peptides generated by limited trypsic digestion, chymotrypsic digestion and N-bromosuccinimide cleavage of the protein. The amino acid sequence so obtained is considered in comparison with those know for other parvalbumins and for rabbit troponin-C. [ABSTRACT FROM AUTHOR]

Exerting control over the size, morphology, and complexity of metal and semiconductor nanoparticles and nanostructures is a requisite for exploring novel phenomena, and the potential applications of these nanomaterials. Bottom-up colloidal chemistry syntheses can benefit from using biomolecules as active elements to influence the formation of inorganic nanoparticles. In this review, we will discuss how three main biomolecule types, (namely DNA; amino acids, peptides, and proteins; and enzymes), can affect the growth of metal and semiconductor nanoparticles. We will present and discuss the templating and non-templating roles of those biomolecules, featuring key aspects and prospects of biomolecule-assisted metal and semiconductor nanoparticle growth. [ABSTRACT FROM AUTHOR]

Although the formation mechanism of biogenically metallic nanoparticles is broadly associated to enzyme mediation, major attention has been given to the role of proteins and peptides in oxido‐reduction of metallic ions leading to these nanostructures. Among the wide range of biomolecules that can act not only as capping agents but also as non‐enzymatic agents to form nanoparticles, disulphide bridge‐containing peptides and amino acids particularly stand out. The literature proposes that they actively participate in the process of nanoparticles' synthesis, with thiols groups and disulphide bridge moieties as the reaction catalytic sites. Similarly, denaturated enzymes containing exposed S–S or S–H moieties are also able to reduce metallic ions to form nanoparticles. This mini‐review is focused on the biogenic synthesis of metallic nanoparticles such as gold, silver, copper, platinum, palladium, lead and selenium, in which proteins, peptides, reductases and even oxido‐reductases act as non‐enzymatic catalysts of the reduction reaction, opening economically and ecologically favourable perspectives in the nanoparticles synthesis field. [ABSTRACT FROM AUTHOR]

The molecular mechanism by which dual-specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G-proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady-state kinetic assays and time-resolved Fourier-transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1IP4BP and RASAL is mediated by the extra GTPase-activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch-II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re-orientation requires specific interactions between switch-II of Rap and helix-α6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different. [ABSTRACT FROM AUTHOR]

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain-containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud. [ABSTRACT FROM AUTHOR]

Proteins and insolubles were recovered from whole Antarctic krill via novel isoelectric solubilization/precipitation using different pH treatments. The protein recovery yield was 45% to 50% (dry basis). The recovered proteins had higher ( P < 0.05) content of essential amino acids (EAAs) and non-EAAs as well as higher ( P < 0.05) ratio of total EAA/total AA than whole krill. The EAAs constituted almost 50% of total AAs. The least extreme pH treatments (pHs 3 and 12) yielded highest ( P < 0.05) content of EAAs. The quality of recovered proteins was high based on EAAs meeting FAO/WHO/UNU recommendations for adults and infants. The basic pH yielded proteins with the lowest ( P < 0.05) amount of minerals and the highest ( P < 0.05) amount of Ca, P, and Mg in the insolubles when compared to the acidic treatments. However, both basic and acidic treatments effectively removed minerals from recovered proteins without the removal of the exoskeleton before processing. Therefore, besides high-quality proteins, the insolubles may provide a mineral supplement in the animal diet. [ABSTRACT FROM AUTHOR]

We previously found that selective restriction of amino acids inhibits invasion of two androgen-independent human prostate cancer cell lines, DU145 and PC3. Here we show that the restriction of tyrosine (Tyr) and phenylalanine (Phe), methionine (Met) or glutamine (Gln) modulates the activity of G proteins and affects the balance between two actin-binding proteins, cofilin and profilin, in these two cell lines. Selective amino acid restriction differentially reduces G protein binding to GTP in DU145 cells. Tyr/Phe deprivation reduces the amount of Rho-GTP and Rac1-GTP. Met deprivation reduces the amount of Ras-GTP and Rho-GTP, and Gln deprivation decreases Ras-GTP, Rac-GTP, and Cdc42-GTP. Restriction of these amino acids increases the amount of profilin, cofilin and phosphorylation of cofilin-Ser3. Increased PAK1 expression and phosphorylation of PAK1-Thr423, and Ser199/204 are consistent with the increased phosphorylation of LIMK1-Thr508. In PC3 cells, Tyr/Phe or Gln deprivation reduces the amount of Ras-GTP, and all of the examined amino acid restrictions reduce the amount of profilin. PAK1, LIMK1 and cofilin are not significantly altered. These data reveal that specific amino acid deprivation differentially affects actin dynamics in DU145 and PC3. Modulation on Rho, Rac, PAK1, and LIMK1 likely alter the balance between cofilin and profilin in DU145 cells. In contrast, profilin is inhibited in PC3 cells. These effects modulate directionality and motility to inhibit invasion. J. Cell. Physiol. 217: 184–193, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

This study investigated the functional impact ofO-linked glycosylation of von Willebrand Factor (VWF) A1 domains on the interaction with platelet receptors. Native or mutant VWF-A1-domains were transiently overexpressed on COS-7 cells as membrane glycosylphosphatidylinositol (GPI)-anchored FLAG-tagged fusion proteins. Cytofluometric analysis assured comparable levels of A1-domain expression among native and mutant homologues as well as for different culture conditions. Expressing native VWF-A1-domains underO-linked glycosylation blocking conditions increased the platelet aggregatory responses observed for fully glycosylated forms. Utilizing a neuronal network for prediction ofO-linked glycosylation of mammalian proteins, threonine (T) and serine (S) residues located in the VWF-A1-loop flanking regions– not in the loop itself– were determined to be glycosylated n-terminal at amino acids T485, S490, T492 and T493 and c-terminal at T705. Simultaneous selective charge-to-alanine mutation of S490, T492 and T493 led to gain in aggregatory responses. When compared with native forms, equivalent alterations of T485 did not dictate functional differences. Any alanine-substitution for T705 revealed a substantial loss in aggregatory effects– possibly as a result of structural desintegration of the VWF-A1-binding site for glycoprotein (GP) Ib. These data suggest specificO-linked glycosylation of the amino-terminal VWF-A1-loop-flanking region to have a negative regulatory impact on the A1-domain affinity of non-activated human VWF for human platelet-GPIb. [ABSTRACT FROM AUTHOR]

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp,Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. TheL. vannameihistone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition againstMicrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 µmwhile a mixture of histones H2B and H4 was active at 3 µm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought. [ABSTRACT FROM AUTHOR]

The HbpR protein is theσ54-dependent transcription activator for 2-hydroxybiphenyl degradation inPseudomonas azelaica. The ability of HbpR and XylR, which share 35% amino acid sequence identity, to cross-activate the P hbpC and P u promoters was investigated by determining HbpR- or XylR-mediated luciferase expression and by DNA binding assays. XylR measurably activated the P hbpC promoter in the presence of the effector m-xylene, both inEscherichia coliandPseudomonas putida. HbpR weakly stimulated the P u promoter inE. colibut not inP. azelaica. Poor HbpR-dependent activation from P u was caused by a weak binding to the operator region. To create promoters efficiently activated by both regulators, the HbpR binding sites on P hbpC were gradually changed into the XylR binding sites of P u by site-directed mutagenesis. Inducible luciferase expression from mutated promoters was tested inE. colion a two plasmid system, and from mono copy gene fusions inP. azelaicaandP. putida. Some mutants were efficiently activated by both HbpR and XylR, showing that promoters can be created which are permissive for both regulators. Others achieved a higher XylR-dependent transcription than from P u itself. Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild-type P hbpC, while keeping the same maximal induction level. On the basis of these results, a dual-responsive bioreporter strain ofP. azelaicawas created, containing both XylR and HbpR, and activating luciferase expression from the same single promoter independently withm-xylene and 2-hydroxybiphenyl. [ABSTRACT FROM AUTHOR]

A novel tripeptide motif called a nest has recently been described in proteins with the function of binding anionic, or partially anionic, atoms such as carbonyl 0 atoms. In the present work, a search for nests in small polypeptides stored in the Cambridge Structural Database is reported. 37 unique examples were found: over half form part of hydrogen-bond arrangements resembling those in proteins, such as Schellman/ paperclip loop motifs, various types of β-turn and Asx-turns or Ser/Thr-turns, while a third are in novel situations, some involving binding to anionic groups from other molecules within the crystal complex. An example is the antibiotic vancomycin, which incorporates a prominent nest forming part of a peptide-binding site. This nest binds the carboxylate of the C-terminal D-alanine of the bacterial cell-wall precursor peptide, thereby inhibiting the final step of bacterial cell-wall synthesis. As in proteins, a number of nests occur in short peptides with an alternating glycine/L-amino- acid sequence but, uniquely to non-ribosomally synthesized short peptides, several nests within them are constructed from alternating D- and L-amino acids, and such sequences seem to specially favour nests. [ABSTRACT FROM AUTHOR]

Focuses on the study which shows that lysine acetylation has been shown to occur in many protein targets. Mechanisms used by lysine acetylation; Docking sites for bromodomain proteins; Relationship between acetyllysine and bromodomains.

A candidate human imidazoline-1 receptor, designated imidazoline receptor antisera-selected (IRAS) protein, was cloned based on immunoreactivity with antiserum against a purified imidazoline receptor binding peptide (IRBP antiserum). Human IRAS is 167 kD in size, different from 33- to 85-kD IRBP bands previously linked to the human platelet I1 receptor. To explore the possible relationship between IRAS and these smaller proteins, seven different epitope-specific antisera against IRAS were raised in rabbits for comparison with IRBP antiserum. Focus was on antiserum227-241, corresponding to amino acids #227 to 241 in IRAS, because this antiserum was found uniquely able to immunoprecipitate non-denatured 85-kD and 170-kD forms of IRAS from a human megakaryoblastoma cell line (MEG01), a model of platelet-producing cells. Human platelets lacked the 170-kD form of IRAS, but 33-kD and 85-kD bands were detectable and seemed to be possible fragments of full-length IRAS. The intensity of the 85-kD band detected by antiserum227-241 was significantly correlated (r 5 0.62, P 5 0.04) with the intensity of the 33-kD band across 11 human platelet samples. A positive correlation between the intensities of the 33-kD and 85-kD bands is consistent with both being fragments of IRAS. [ABSTRACT FROM AUTHOR]

The human I1-imidazoline receptor candidate gene, iras, has previously been cloned and mapped to locus 3p21.1-9 (also known as Nischarin; accession #AC006208). By comparison to a database of expressed sequence tags (ESTs), three alternatively spliced transcripts have been deduced. A map of 21 exons was constructed for the medium-length transcript (IRAS-M) containing 5,232 base pairs (bp) and encoding 1,504 amino acids (aas). Introns 13B and 13C are inserted into the two alternative transcripts, forming IRAS-S and IRAS-L mRNA (short and long isoforms). Northern blots confirmed the existence of these mRNA isoforms. In most brain regions the order of mRNA abundance was IRAS-M > IRAS-L > IRAS-S mRNA. Although aas 1 through 510 are theoretically identical, truncated proteins could be derived from IRAS-S (2,678 bp transcript yields 515 aas) and IRAS-L (9,457 bp transcript yields 583 aas). Because exon-16 of the iras gene has been proposed to encode the functional domains of imidazoline and a-5 integrin binding, only IRAS-M is expected to possess I1 receptor properties. Subtype-selective cDNA expression constructs were therefore generated and used to transfect CHO cells. High-affinity I1 binding was endowed by IRAS-M and IRAS-L, but not by IRAS-S transfection. [ABSTRACT FROM AUTHOR]

Presents a study that proposed a laboratory exercise in biochemistry to guide students to the comprehension of basic aspects in determining structure and physicochemical properties of proteins. Amino acid composition of human proteins and silk fibroin; Flow chart for fractionation of plasma proteins by precipitation with ammonium sulfate; Relative dimensions of human proteins.

Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation. [ABSTRACT FROM AUTHOR]

A Sin3-interacting domain (SID) originally described in Mad proteins is necessary for both transcriptional repression and growth suppression by these transcription factors. We recently reported that a structurally and functionally related Mad1-like SID is also present in five Sp1-like repressor proteins (TIEG1, TIEG2, BTEB1, BTEB3 and BTEB4), demonstrating that SID-mSin3A interactions have a wider functional impact on transcriptional repression. SID-mSin3A interaction is necessary for the anti-proliferative function of Mad, TIEG and BTEB proteins. It remains to be established, however, whether the SID-mSin3A interaction is constitutive or regulated. Here, we describe that the Mad1-like SID domain of the Sp1-like repressor TIEG2 is inhibited by the epidermal growth factor (EGF)-Ras-MEK1-ERK2 signaling pathway, via phosphorylation of four serine/threonine sites adjacent to the SID. This phenomenon disrupts the SID-mSin3A interaction and thereby inhibits TIEG2's repression activity. Thus, these results show for the first time that the repression of a SID-containing protein is regulated by signaling rather than functioning in a constitutive manner, extending our understanding of how the function of SID-containing repressors may be controlled. [ABSTRACT FROM AUTHOR]

11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGα activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGγ only activates cleavage after basic residues. We have isolated REGγ mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome β subunits. The most robust REGγ specificity mutants involve substitution of Glu or Asp for Lys188. REGγ(K188ELD) variants are virtually identical to REGα in proteasome activation but assemble into less stable hepta- mers/hexamers. Based on the REGα crystal structure, Lys188 of REGγ faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin- like subunits by 125I-YL3-VS demonstrates that REGγ(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGγ(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGγ molecule. [ABSTRACT FROM AUTHOR]

The presence of endoproteinases in the intercellular washing fluid of the first wheat (Triticum aestivum) leaf 13 days after sowing was investigated. Two activities were detected after separation of the intercellular fluid proteins by SDS-PAGE using slab gels containing gelatin as substrate. Their sizes were 70 and 100 kDa. Both enzymes hydrolyzed gelatin and casein. They also seem to be involved in the degradation of a 40 kDa protein component of the intercellular fluid. These results indicate that both enzymes are endoproteinases. They were also obtained from leaves previously submitted to intercellular fluid extraction. However, the 100 kDa enzyme yield was low, indicating that it is mainly located in the intercellular washing fluid. Assays performed with either specific substrates or inhibitors indicate that both enzymes are serine proteinases. [ABSTRACT FROM AUTHOR]

Autumnal changes in total nitrogen, salt‐extractable protein and amino acid concentrations in leaves and adjacent bark of black alder [Ainus glutinosa (L.) Gaertn.], eastern cottonwood (Populus deltoides Bartr. ex Marsh.) and white basswood (Tilia heterophylla Vent.) were determined for trees growing on minespoils and a prairiederived loamy soil in central Illinois. The composition of free amino acids in foliage was also determined at peak concentration for each tree species during late senescence. Total nitrogen concentration in the leaves decreased slowly throughout most of the fall for all species. In the final stages of senescence, total leaf nitrogen concentrations were about halved in eastern cottonwood and white basswood but continued to decrease slowly in black alder. The concentration of salt‐extractable proteins in leaves of all species peaked early in the fall and then declined prior to leaf abscission. This decline coincided with an increase in the concentration of free amino acids in the leaves. The increase stabilized in both eastern poplar and white basswood but continued in black alder. Glutamine in black alder and eastern cottonwood, and asparagine in white basswood were the most abundant free amino acids at the time of peak concentration of total free amino acids in senescent leaves. Bark of trees of all species had higher nitrogen concentrations and higher proportions of salt‐extractable proteins to estimated total proteins after leaf senescence than during the preceding summer. Results indicate that autumnal fluxes in leaf and bark nitrogen fractions of alder can differ substantially from fluxes in other broadleaved winter‐deciduous trees in a way which suggests that alder does not effectively conserve leaf nitrogen through retranslocation to bark tissue. [ABSTRACT FROM AUTHOR]

P pili are composite adhesive fibres that allow uropathogenic Escherichia coli to gain a foothold in the host by binding to receptors present on the uroepitheltum via the adhestn PapG. The assembly of P pill requires a periplasmic chaperone, PapD, that has an immunoglobulin-like three-dimensional structure. PapD--subunit complex formation involves a conserved anchoring mechanism in the chaperone cleft and a 'molecular zippering' to the extreme C-terminus of pilus subunits. A chaperone-binding assay was developed using fusions of the C-terminus of PapG to maltose-binding protein (MBP/G fusions) to investigate whether chaperone-subunit complex formation requires additional interactions. PapD bound strongly to an MBPIG fusion containing the C-terminal 140 amino acids of PapG (MBP/G175-314) but only weakly to the MBP/G234-314 fusion containing 81 C-terminal residues, arguing that the region between residues 175-234 contains additional information that is required for strong PapD-PapG interactions. PapD was shown to interact with a PapG C-terminal truncate containing residues 1-198 but not a truncate containing residues 1-145, suggesting the presence of a second, independent PapD interactive site. Four peptides overlapping the second site region were tested for binding to PapD in vitro to further delineate this motif. Only one of the peptides synthesized was recognized by PapD. The MBP/G fusion containing both binding sites formed a tight complex with PapD in vivo and inhibited pilus assembly by preventing chaperone--subunit complex formation. [ABSTRACT FROM AUTHOR]

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185–219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coti RecA in this region did not. The binding of RecA to ssDNA examined using a gel-shill assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301–329 of the C-terminus or from N-terminal residues 6–39. A peptide corresponding to amino acid positions 152–169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 μM. We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193–212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185–224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303–353). In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193–212 is either part of or the whole ssDNA-binding domain of the RecA protein. [ABSTRACT FROM AUTHOR]

A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL-6B chromatography and Mono-S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. They were referred to as p1, p2 and p3, according to their elution order from the Mono-S column. Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7-kDa DNA-binding proteins previously isolated in Sulfolobus strains [Choli, T., Wittman-Liebold, B. & Reinhardt, R. (1988) J. Biol. Chem. 263, 7087-7093]. The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE. Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure. Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7 - 7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate. The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity. This was also confirmed by incubating p2 with tRNAfMet Met (fMet, N-formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3'-phosphooligonucleotides. [ABSTRACT FROM AUTHOR]

The bovine serum amyloid A (SAA) and tissue amyloid A (AA) proteins were isolated and characterized. SAA was isolated from acute phase high density lipoprotein (HDL) of a cow suffering from acute mastitis, and was identified by amino acid sequence analysis. No AA-like protein was found in complex with HDL in serum. Amyloid fibrils isolated from a bovine kidney contained a 9 kDa AA protein and a considerable amount of a 14 kDa protein. Amino acid sequence analysis showed that the largest protein probably represents undegraded SAA. This is an interesting observation which confirms previous works indicating that SAA can be incorporated in the amyloid fibrils without a prior degradation to AA. The partial amino acid sequences of bovine SAA and AA were strikingly homologous to the sequences of corresponding proteins in man and other species. [ABSTRACT FROM AUTHOR]

Proteins from the large and small subunits of Aedes alhopictus (mosquito) cytoplasmic ribosomes were characterized by two-dimensional polyacrylamide gel electrophoresis. The small subunit contained 28 - 31 proteins ranging in molecular mass from 10 to 49 kDa. The large subunit contained 36-39 proteins that ranged in molecular mass from 11 to 53 kDa. The largest protein on the small subunit, S1, was the predominant phosphorylated ribosomal protein. Under long-term labelling conditions, L4 and L33 were also phosphorylated. Peptide mapping by partial proteolysis indicated that Ae albopictus S1 may share partial amino acid homology with the phosphorylated ribosomal protein S6 from Drosophila melanogaster. Unlike Drosophila $6, however. Aedes S1 was not dephosphorylated during heat shock. Treatment of mosquito cells with the insect molting hormone 20-hydroxyecdysone did not affect phosphorylation of ribosomal proteins. [ABSTRACT FROM AUTHOR]

Gel filtration of Peking duck eye lens proteins reveals a component eluting just behind δ-crystallin and comprising approximately 10% of the total soluble protein. The native Mr of this additional component is estimated to be 120000; it appears to be composed of three identical chains of Mr 38000 and pI 7.5. Circular dichroic spectroscopy showed a relatively high α-helical content. No immunological cross-reactivity is found with α-, β,- γ- or δ-crystallins, and partial amino acid sequence determinations likewise failed to reveal any similarity with other known crystallins. We conclude that this protein represents another and novel family of eye lens proteins, for which we propose the designation ε-crystallin. ε-Crystallin is translated from a 1450-base mRNA, which has been partially purified. ε-Crystallin is found scattered among avian and reptilian taxa, but not in other vertebrates. Its rate of evolutionary change seems to be as slow as that of α- and β-crystallins. [ABSTRACT FROM AUTHOR]

Core particles prepared by micrococcal nuclease digestion of pig liver chromatin have been adsorbed on hydroxyapatite and dissociated by gradual increase in ionic strength and finally by urea and guanidine. By this method non-histone proteins have been found to be associated with the core particles. Proteins tightly bound to the core particle DNA (i.e. dissociated only by urea and guanidine) have also been found: these are proteins with a limited heterogeneity, with respect to their molecular weights, since only six components are present with molecular weights ranging from 71000 to 20000. They show, furthermore, a peculiar amino acid composition. Other tightly bound proteins have been shown to be present only in the spacer regions. The existence of two different classes of tightly bound proteins probably reflects different modes of binding to the DNA, which are come patible or incompatible, respectively, with the simultaneous binding of the histone octamer. [ABSTRACT FROM AUTHOR]

The carbohydrate structure and complete amino acid sequence of a human lambda-type immunoglobulin light chain, protein Sm Z, has been determined. The protein was isolated from the urine of a patient with a plasma cell dyscrasia resembling γ-heavy-chain disease. 13 tryptic peptides covering the entire polypeptide chain of 135 residues were isolated from the aminoethylated protein, and 15 chymotryptic peptides, accounting for 131 residues, were recovered from the carboxymethylated protein. The sequence of 18 of these peptides was partially or completely determined by the Edman-dansyl technique or C-terminal analysis, permitting the establishment of the complete primary structure of the polypeptide chain. The sequence established that this light chain possessed an intramolecular deletion of 81 amino acid residues. The N-terminal 30 residues showed considerable homology with other lambda chains of subgroup II. The defect began at position 31, in the first hyper-variable region, and encompassed the remainder of the variable region through position 109. The constant region was fully intact and normal synthesis recommenced with a glutaminyl residue at position 110, the first residue of the constant region. This light chain contained carbohydrate in the hyperyanable region just preceding the deletion. The precise number and locations of the oligosaccharide chains were established by amino acid sequence analysis of glycopeptides isolated from proteolytic hydrolysates by chromatography on Bio-GeI P-6 columns. These studies showed that protein Sm A contains one N-glycosidically-linked chain attached to asparagine-25 and one O-glycosidically-linked oligosaccharide chain attached to serine-21. The structures of the oligo-saccharide chains were determined by methylation analysis, gas chromatography and hydrolysis with specific glycosidases. The structure of the N-glycosidically-linked chain was NeuAc(α2-→6)Gal(β1→4)GlcNAc(β1→2)- Man(α1→6)[NeuAc(x2→6)Gal(β→4)GlcNAc(β1→2)Man(α1→3)]Man(β1→4)GlcNAc(β1→4)[Fucα1→6]-GlcNAc→Asn. The second O-glycosidicalIy-linked chain was a disialylated tetrasaccharide with the structure, Neu(x2→3)Gal(β1→3)[NeuAc(α2→6)GalNAC→Ser. This mucin-type disialylated tetrasaccharide in close proximity to N-asparagine-linked chains has not been previously observed in the oligosaccharide chains of immunoglobulins. [ABSTRACT FROM AUTHOR]

The N-trifluoroacetyl-L-prolyl-leucyl methyl ester (TFA-L- prolyl-leucine) was synthesized to quantitate the amounts of D- and L-leucine in the amino acid pool fraction of tobacco callus tissue. TFA-L-prolyl-D-leucine and TFA-L-prolyl-L-leucine could be separated on gas liquid chromatography, with the former eluting about 45 s before the latter. The chromatographed TFA-L-prolyl-L-leucine was collected from the gas liquid chromatograph and found to contain equal molar amounts of leucine and proline. The identity of the two gas liquid chromatography peaks was confirmed by gas liquid chromatography/mass spectrometry. Although callus tissue incorporated both exogenously added D- and L-leucine into its amino acid pool fraction, the rate of uptake of D-leucine appeared to be substantially less than that of L-leucine. When callus tissue was labeled on ³H-L-leucine medium for 24 h and then transferred to nonradioactive medium for 24 h, about 50% of the tritium remaining in the leucine pool was not available for protein synthesis (nonprecursor pool fraction). However, all of the tritium was associated with L-leucine, and the specific radioactivity of leucine in collected TFA-L-prolyl-leucine was the same as that of leucine in the crude pool fraction. Thus, the nonprecursor pool is L-leucine. [ABSTRACT FROM AUTHOR]

Two mutants of Chlorella vulgaris characterized by high (HS) or low (LS) content of sulphur‐amino acids were compared to the wild strain with respect to their protein electrophoretic pattern. Three new bands, not present in the LS and wild strains, were found in the HS mutant. In algae grown in the presence of 35S labelled sulphate two of these new bands showed a very high 35S specific activity. Some electrophoretic bands common to LS, HS, and wild strain, were present in different proportions and showed different levels of specific radioactivity for each strain. Therefore, mutational events appear to have affected both the amino acid composition of single proteins and the relative amount of proteins with high and low sulphate content. [ABSTRACT FROM AUTHOR]