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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]

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]

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]

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]