The formation of the triple helix in a mixed solution of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n'. A model of molecular size recognition

A theoretical analysis is given of the triple-helix–random-coil transition in a mixed solution of poly(Pro-Pro-Gly)_n with two different but defined degrees of polymerization n and n′. Because of the highly cooperative nature of this helix–coil transition, each polypeptide chain tends to form a triple helix with other polypeptide chains with the same degree of polymerization (size recognition). Occurrence of triple helices consisting of polypeptide chains with different degrees of polymerization (error in recognition) is studied in detail as a function of the cooperativity, and n and n′. Implication of this analysis for molecular recognition in general is discussed.     

Conformational change of the triple-helical structure. II. Conformation of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n (Ala-Pro-Gly)m(Pro-Pro-Pro-Gly)n in an aqueous solution

Measurements of the molecular weight of (Pro-Pro-Gly)_n and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n, which were synthesized by the solid-phase method, revealed that they formed a trimer in an aqueous solution, and dissociated into single-stranded chains on warming. Accompanying the transition, a large decrease of optical rotation was observed, like the collagen–gelatin transition. The shape of the trimeric molecule was rodlike, and the dimensions were 12 Å in diameter and 2.8 Å per residue in length, regardless of the length of Ala-Pro-Gly sequences in a peptide chain. The data indicate that both Pro-Pro-Gly sequences and Ala-Pro-Gly sequences from the triple-helical structure similar to that of collagen in aqueous solution. All optical rotational dispersion (ORD) curves of solutions of the peptides were represented by a single-term Drude equation, and the Drude constant λ_c was 200 nm for all peptides regardless of the length of Ala-Pro-Gly sequences. The resemblance between the helical structure formed by Pro-Pro-Gly sequences and that by Ala-Pro-Gly sequences was also suggested by the formation of the hybrid triple helix from two kinds of peptide chains with different lengths of Ala-Pro-Gly sequences.     

Conformational change of the triple-helical structure. I. Synthesis of model peptides of collagen by the solid-phase method

As model peptides of collagen, (Pro-Pro-Gly)_n (n = 10, 12, 14, and 15) and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n (2n + m = 15; m = 1, 3, and 5) were synthesized by the solid-phase method. The final products were pure when checked by high-voltage paper electrophoresis and by amino acid analysis. Elemental composition was also examined.     

The association reaction of collagen model polypeptides (Pro-Pro-Gly)n

The characterization of recently synthesized (Pro-Pro-Gly)_n, n = 7, 8 is described, along with melting profile studies of its association equilibrium, and thermal quenching studies of the kinetics of its association reaction. The order of the kinetic reaction is about 3, implying that three peptide chains are involved in the activated state of the rate-limiting step. The reaction rate was found to exhibit a negative temperature coefficient. With the (Pro-Pro-Gly)₇ peptide, the concentration dependence of the (Pro-Pro-Gly)_n association equilibrium was observed for the first time. Detailed thermodynamic analysis for these n = 7, 8 data, together with literature data for n = 10, 15, 20 were carried out for both the simple “all-or-none” binding model and for a series of complex equilibrium models. For the latter, all of the (Pro-Pro-Gly)_n data (in 10% acetic acid) are fit best with a maximally cooperative near-neighbor model with a standard enthalpy change ΔH = ?650 cal/mole of residues, and a standard entropy change ΔS = ?14.63 ?10/n cal/deg-mole of residues, wherein the ?10 eu represents an end-effect contribution to the binding free energy. With regard to optical rotatory properties and thermodynamic parameters, the data for the new n = 7, 8 peptides match rather well with the literature data for the n = 10, 15, and 20 peptides. The enthalpic stabilization per residue of the triple-helical form of (Pro-Pro-Gly)_n was nearly an order of magnitude smaller than the enthalpic stabilization per additional proline obtained from direct calorimetric measurements on native collagens of different (and much lower) proline contents by Privalov and Tiktopulo. [Biopolymers (1970) 9 , 127–139.] Possible explanations for this phenomenon are discussed.     

Physicochemical analysis of (Pro-Pro-Gly)n with defined molecular weight--temperature dependence of molecular weight in aqueous solution

Sequenced polytripeptides, (Pro-Pro-Gly)_n, (n = 10, 15, 20), with defined molecular weights were synthesized by the solid-phase method. Conformational changes of these sample as a function of temperature were studied by measurements of optical rotation and sedimentation equilibrium. The temperature dependence of optical rotation was shown similar to thermal transition of collagen molecule. Each of these polymers existed as a timer at lower temperature. (Pro-Pro-Gly)₁₀ existed as a monomer at higher temperature, and the others were expected to behave analogously.     

Conformational change of the triple-helical structure. III. Stabilizing forces in the triple helix

The analysis of thermal melting curves of (PPG)_n (n = 10, 12, 14, and 15) and (PPG)_n(APG)_m (PPG)_n (2n + m = 15; m = 1, 3, and 5) revealed that the enthalpy and entropy changes accompanying the transition from the random coil to the triple helix are ?2500 cal and ?6.3 e.u. per one mole of the tripeptide of the form of Pro-Pro-Gly, and ?3100 cal and ?11.2 e.u. per one mole of the tripeptide of the form of Ala-Pro-Gly. The thermal instability of the triple helix composed of Ala-Pro-Gly sequences, compared to the helix of Pro-Pro-Gly sequences, is due to the larger entropy change of Ala-Pro-Gly (?11.2 e.u.) compared to that of Pro-Pro-Gly (?6.3 e.u.), not from the difference in the enthalpy change. The difference in the enthalpy change between Pro-Pro-Gly and Ala-Pro-Gly arises from the hydrophobic bond between two pyrrolidine rings of proline residues formed in the triple helix. Since the enthalpy change for the formation of hydrophobic bonds is positive, it is also concluded that only one hydrogen bond is formed in a tripeptide unit, regardless of the amino acid sequence. The enthalpy change for the formation of this hydrogen bond is ?3100 cal/mol, and that of the hydrophobic bond between two pyrrolidine rings is +600 cal/mol.     

The enzymic hydroxylation of protocollagen models

1. Synthetic polymers of l-prolyl-l-prolylglycine of defined chain length, (Pro-Pro-Gly)_n, were found to be substrates for the enzyme protocollagen–proline hydroxylase, with optimum chain length n=5. Boiling the polymer (Pro-Pro-Gly)₁₅ increased its activity as a substrate but had no effect on (Pro-Pro-Gly)₅. 2. Protection of both or one of the N- and C-terminal groups made (Pro-Pro-Gly)₃ a better substrate, and collagenase digestion of hydroxylated tert.-pentyloxy-carbonyl-(Pro-Pro-Gly)₃ benzyl ester indicated that the central prolyl residues were the major points of hydroxylation. 3. The results suggest that the long-chain peptides are optimum substrates but that a triple-stranded structure is inhibitory for hydroxylation.     

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

We have synthesized (Pro-Pro-βAla)_n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)_n followed a single-term Drude equation and the λ_c was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)_n to (Pro-Pro-Gly)_n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.     

Abundance and degree of dispersion of genomic d(GA) n ·d(TC) n sequences

Summary The abundance of d(GA) _n ·d(TC) _n tracts was determined in genomes of rodents and primates. Dot blot hybridization assays revealed that such tracts constitute 0.40%, 0.30%, and 0.40%, respectively, of the rat, hamster, and mouse genomes, but only 0.07% and 0.05% of the human and monkey genomes. A plaque hybridization assay of rat and human genomic libraries showed that 37% and 16%, respectively, of the recombinant phages in these libraries contain d(GA) _n ·d(TC) _n tracts. A survey of sequences stored in the GenBank data bank showed that a significant fraction of the stored rodent genes (about 2.0%) contain long d(GA) _n ·d(TC) _n tracts (n> 30) with <10% mismatching. The primate genes contain only shorter tracts (n<15) with <10% mismatching. In addition, the rodent and the primate genes contain tracts with larger degrees of mismatching. The chicken, which represents an entirely different branch of the evolutionary tree, was found to be as low in d(GA) _n ·d(TC) _n tracts as the primates. It is suggested that a common ancestor of the rodents has acquired the ability to amplify d(GA) _n ·d(TC) _n tracts.     

Block copolymers of amino acids. I. Synthesis and structure of copolymers of L-alanine or L-phenylalanine with D,L-lysine-d7 or D,L-lysine

Water-soluble block copolymers of the type (A)_m-(B)_n-(A)_p, where (A)_m,p was either poly(D ,L -lysine-α,β,β,γ,γ,δ,δ-d₇) or poly(D ,L -lysine) and (B)_n was either poly(L -alanine) or poly(L -phenylalanine), were synthesized for conformational studies by proton magnetic resonance spectroscopy. Analytical determination of the amount of the initiator fragment (n-hexylamine) at the C-terminus of the copolymers was used to obtain the number-average degrees of polymerization, DP _n, and thereby, together with the amino acid composition, to establish the covalent structures of the polymers. The values of DP _n were found to be much lower than those deduced from sedimentation equilibrium or form viscosity measurements. These deviations, which also are thought to have arisen in similar studies reported in the literature, are attributable to intermolecular aggregation; the relation of such aggregation to covalent structure (and its effect on the polymerization reaction) is discussed in terms of the conditions and mechanism of synthesis of block copolymers of amino acids.     

DNA fingerprinting in horses using a simple (TG)n probe and its application to population comparisons

A synthetic polynucleotide (TG)_n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 times 10-⁴-7 times 10-⁶ The variability derived with the (TG)_n, probe in horses was higher than what we obtained with several other commmonly used probes for DNA fingerprinting. Individuals within breeds tended to be more similar to each other with regard to DNA fingerprint pattern than to individuals of other breeds. Moreover, a parsimony analysis made on the basis of the hybridization patterns gave clustering of individuals within breeds. The possibility of using hypervariable probes for the identification of breed-specific characters is discussed.     

Measurement of vitamin K in human liver by gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection

A sensitive and precise method for measuring endogenous phylloquinone (K₁) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K₁ and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n=10) and cirrhosis (n=22) were significantly lower than in normal livers (n=6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.     

Chromosomal Location of Ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n Telomeric Repeats in the Neogastropod Fasciolaria Lignaria (Mollusca: Prosobranchia)

This paper reports on a successful application of fluorescent in situhybridization (FISH) with three repetitive DNA probes (ribosomal DNA (rDNA), (GATA)_nand (TTAGGG)_n) in the chromosomes of Fasciolaria lignaria(Mollusca: Prosobranchia: Neogastropoda). rDNA FISH consistently identified four chromosome pairs per spread in the three examined specimens. The telomeric sequence (TTAGGG)_nhybridized with termini of all chromosomes. GATA FISH revealed abundant, dispersed minisatellite regions which were not associated to the XY sex-determining mechanism as indicated by the absence of a Y specific pattern of labelling. This revised version was published online in July 2006 with corrections to the Cover Date.     

Block oligopeptides (L-lysyl)m-(L-alanyl) L-tyrosyl-(L-alanyl)n- (L-lysyl)m. I. Synthesis through fragment condensation and characterization

Model peptides containing one aromatic residue were synthesized and characterized in order to investigate their interactions with polynucleotides. Chromatographically pure block oligopeptides (L -alysyl)_m-(L -alanyl)_n- L -tyrosyl- (L -alanyl)_n, with n = 3 and m=3 or 6, were prepared by fragments condensation using the mixed anhydride method. The protected fragments were prepared by stepwise addition of amino acid residues through the dicyclohexylcarbodiimide method. The purity of the intermediate coupling product was analyzed by gradient elution chromotography on carboxylmethylcellulose. Both block oligopeptides were isolated by preparative chromatography on carboxymethylcellulose. The different features of these syntheses are discussed.     

Intra- and intermolecular charge-transfer interactions between terminal electron donor and acceptor attached to poly(γ-benzyl-L-glutamate) in solution

Poly(γ-benzyl-L -glutamate) having a terminal dimethylaminoanilide group as an electron donor (D) and a terminal 3,5-dinitrobenzoyl group as an electron acceptor (A) (A-[Glu(OBzl)]_n-D) was synthesized by the N-carboxyanhydride method. Polymer samples were fractionated by gel chromatography and their number-average degrees of polymerization n were determined by the absorbances of the terminal chromophores. These polymers in chloroform and dimethylformamide solutions showed a charge-transfer (CT) absorption band around 455 nm, and the fraction of the polymer forming the CT complex was evaluated as a function of the chain length. CT absorption for short chains (n = 5 ～ 20) was attributed to intramolecular CT complex in which the A-[Glu(OBzl)]_n-D chain takes cyclic conformations. An optimum chain length for the intramolecular CT was found to be n ? 10, where the [Glu(OBzl)]_n chain may most easily bend back to form cyclic conformations. Stronger CT absorption observed for longer chains than n = 20 was shown to be intermolecular, and an intermolecular head-to-tail aggregation was found to be a cause of the strong CT interaction. All helical A-[Glu(OBzl)]_n-D chains were found to form the head-to-tail dimers in chloroform solution.     

Method for Enrichment of Rhodopseudomonas sphaeroides from Fresh Water

An inulinase was highly purified from the culture broth of Penicillium purpurogenum by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-65, and Bio-Gel P-100. The enzyme was homogeneous by disc electrophoretic analysis. The molecular weight was 6.4 × 10⁴ by SDS-disc electrophoresis and gel filtration on Bio-Gel P-150. The isoelectric point was pH 3.6 by isoelectric focusing. The enzyme hydrolyzed inulin rapidly, but did not affect sucrose. By paper chromatography analysis, the major products from inulin were tri-, tetra-, penta-, and hexa-saccharides. The substrate specificity of the enzyme on hydrolyses of fructo-oligosaccharides[1^F(1-β-d-fructofuranosyl)n sucrose (n = 1 to 6 and n (average of polymerization degree) = 8)] were examined. The Km values and relative maximum velocities for the hydrolyses of inulin and fructo-oligosaccharides (GF_n, n = 2 to 7 and n = 9) were as follows: inulin, (DP = 35) 0.21 mM and 100; GF₉, 0.24 mM and 86.5; GF₇, 0.33 mM and 132; GF₆, 0.85 mM and 71.2; GF₅, 3.8 mM and 25.4; GF₄, 2.8 mM and 28.8; GF₃, (nystose) 16 mM and 0.8; GF₂ (1-kestose), 8.4 mM and 0.2. The molecular activities for the hydrolyses of fructo-oligosaccharides (GF_n, n = 2 to 6) were increased depending on the degree of polymerization of fructosyl residues, and were nearly constant if the polymerization degree was over seven. These results strongly suggested that the endo-type inulinase from Penicillium purpurogenum had a subsite structure consisting of at least seven subsites.     

A reducing-end-acting chitinase from <Emphasis Type="Italic">Vibrio proteolyticus</Emphasis> belonging to glycoside hydrolase family 19

A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus (chi19) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N-acetylated chitooligosaccharides, (GlcNAc) _n , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc) _n at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)₂ from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.     

Restriction fragment polymorphism in the sex-determining region of the Y chromosomal DNA of European wild mice

Summary Using ³²P-labeled probe consisting mainly of (GATA)_n we have shown that a male specific Alu1 DNA blot pattern which defines the Y chromosome sex-determining locus in inbred mice is highly polymorphic in wild mice, indicating substantial sequence evolution in this region under field conditions. In all cases examined by in situ hybridization, the region concerned is paracentromeric. In contrast, the blot pattern of another probe (M 34) which detects repeated sequences specific to the mouse Y chromosome but outside the sex-determining locus, remains constant between different isolates.Deceased     

Early stages of sex chromosome differentiation in fish as analysed by simple repetitive DNA sequences

Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically demonstrable sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)_n/(gaca)_m are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)₄ probe. The (GATA)₄ oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In contrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)₄. Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)₄ probe.by E.R. SchmidtDedicated to Professor Karl Sperling on the occasion of his 50th birthday     

Age-related prevalence of antibodies to infective larvae ofWuchereria bancrofti in normal individuals from a filaria-endemic region

Antibody isotypic levels (IgM, IgE and IgG subclasses) to infective larvae (L₃) ofWuchereria bancrofti were measured in 104 normal individuals from a filaria-endemic region in Orissa. The titres of antibodies were considerably higher in adults (n = 25, 25.1± 3.8 year) than in children (n = 52, 7.1 ± 2.1 year). Young children (n = 14) less than four years were seronegative to all isotypes other than IgM, the sero-conversion to which was achieved in the children (n=15) by the age of 7.5±1.2 years. The prevalence of other isotypes increased with age and reached a maximum in early adulthood (18.6 ±1.6 years), which persisted in older adults (> 30 years). However, the increase in IgG₃ prevalence with age was less marked. IgG₂ was detected only after 10 years of age. Compared to the high prevalence (100%) of IgM, IgE, IgG₁, and IgG₂, in adults. IgG₃ and IgG₄ prevalences were low, 35% and 28% respectively. IgA level to L₃ antigen was found to be extremely low even in adults. These data indicate that the prevalence of L₃ antibodies was different for different isotypes and the acquisition of antibody response essentially followed an age dependent pattern.