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Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-α,α-1,1-glucose) and maltose (glucosyl-α1-4-glucose). TreS was purified from the cytosol ofMycobacterium smegmatisto give a single protein band on SDS gels with a molecular mass of≈ 68 kDa. However, active enzyme exhibited a molecular mass of≈ 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, thetreSgene was identified in theM. smegmatisgenome sequence, and was cloned and expressed in active form inEscherichia coli.The recombinant protein was synthesized with a (His)6 tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42–45% of each) was reached during an incubation of about 6 h, whereas at 2 mmmaltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in≥ 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8–10% free glucose. TheKm for maltose was≈ 10 mm, whereas for trehalose it was≈ 90 mm. Whileβ,β-trehalose, isomaltose (α1,6-glucose disaccharide), kojibiose (α1,2) or cellobiose (β1,4) were not substrates for TreS, nigerose (α1,3-glucose disaccharide) andα,β-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer.[3H]Trehalose is converted to[3H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and[14C]maltose produces[14C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as[3H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of[3H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry. [ABSTRACT FROM AUTHOR]

Biosynthetically directed fractional 13C labeling of proteinogenic amino acids is achieved by expression of proteins on a minimal medium which contains a mixture of [13C6]glucose and glucose with natural isotope abundance as the sole carbon source. Subsequent hydrolysis of the proteins yields the free amino acids. The observation of 13C-13C spin-spin scalar coupling fine structures in sensitive two-dimensional heteronuclear [13C,1H]-correlation spectroscopy (2D [13C,1H]-COSY) allows one to identify non-random 13C-labeling patterns arising from the incorporation of intact two-carbon and three-carbon fragments from a single source molecule of glucose into the amino acids. Since 2D [13C,1H]-COSY suffices to resolve all relevant resonances, the mixture of amino acids can be analyzed without further separation of its components. Probabilistic equations relate the observed multiplet intensities of the 13C fine structures to the relative abundance of the intact carbon fragments. They enable a quantitative analysis of the carbon flux in the network of biosynthetic pathways, thus using the proteinogenic amino acids as probes to study intermediary metabolism. This paper shows that biosynthetically directed fractional 13C labeling of amino acids provides an efficient analytical tool to quantitatively investigate glycolysis, pyruvate metabolism, pentose phosphate pathway, tricarboxylic acid cycle and C1 metabolism. Possible applications of the method include both the exploration of unknown biosynthetic pathways and the rapid elucidation of the response of a known biosynthetic reaction network to changes in growth conditions or genetic manipulations. In conjunction with the relatively low costs for isotopes, manpower and NMR instrument time, this makes biosynthetic fractional 13C labeling of proteinogenic amino acids particularly attractive to support process design and metabolic engineering in biotechnology, since screening procedures become feasible which enable a systematic characterization of the cell's metabolic state as a function of parameters that are involved in the optimization of biotechnological processes.

A protein is generally classified into one of the following four structural classes: all alpha, all beta, alpha+beta and alpha/beta. In this paper, based on the weighting to the 20 constituent amino acids, a new method is proposed for predicting the structural class of a protein according to its amino acid composition. The 20 weighting parameters, which reflect the different properties of the 20 constituent amino acids, have been obtained from a training set of proteins through the linear-programming approach. The rate of correct prediction for a training set of proteins by means of the new method was 100%, whereas the highest rate of previous methods was 82.8%. Furthermore, the results showed that the more numerous training proteins, the more effective the new method.

This paper presents the first purification of the mitochondrial branched‐chain amino acid aminotransferase (BCATm) from sheep placenta. It is a homodimer with an apparent subunit molecular mass of 41 kDa. The enzyme differs from those of the rat and human as it appears to form at least one intermolecular disulfide bond. The sheep BCATm cDNA (1.4 kb) encodes a mature polypeptide of 366 amino acids with a calculated molecular mass of 41 329 Da and a partial mitochondrial targeting sequence of seven amino acids. The sheep BCATm sequence shares higher identity with other mammalian BCATm isoenzymes (82–85%) than with the cytosolic isoenzymes (60%). By Northern blot analysis, a message of 1.7 kb was detected in sheep placenta and skeletal muscle. Measurements of BCAT activity, mRNA and BCATm protein in sheep placenta and skeletal muscle revealed that BCATm is the sole BCAT isoenzyme expressed in placenta, whereas it contributes 57 and 71% of the BCAT activity in tensor fascia latae and masseter muscles from weaned lambs respectively. Skeletal muscle, the main site of branched‐chain amino acid transamination, exhibits significantly lower BCAT activity in sheep than in rat. Our results suggest that the low BCATm mRNA level probably accounts for the low BCAT activity in sheep skeletal muscle, and that the metabolic scheme for branched‐chain amino acid catabolism is specific to each species. [ABSTRACT FROM AUTHOR]

We have previously isolated a form of protein phosphatase-1 (PP1M) from avian smooth muscle myofibrils that is composed of the catalytic subunit of PP1 (PP1C) bound to an M-complex consisting of 110-kDa (M110) and 21-kDa (M21) subunits. The interaction of PP1C with an N-terminal region of the M110 subunit enhances the dephosphorylation of myosin and suppresses the dephosphorylation of other substrates [Alessi, D. R., MacDougall, L. K., Sola, M. M., Ikebe, M. & Cohen, P. (1992) Eur. J. Biochem. 210, 1023–1035; Chen, Y. H., Chen, M. X., Alessi, D. R., Campbell, D. G., Shanahan, C., Cohen, P. & Cohen, P. T. W. (1994) FEBS Lett. 356, 51–56; Johnson, D. F., Moorhead, G., Caudwell, F. B., Cohen, P., Chen, Y. H., Chen, M. X. & Cohen, P. T. W. (1996) Eur. J. Biochem. 239, 317–325]. In this paper, we establish that PP1M accounts for nearly all the myosin phosphatase activity in myofibrils, that the M110 and M21 subunits are present at similar concentrations in the myofibritlar fraction, and that these subunits are entirely bound to PP1. We demonstrate that the M21 subunit does not interact with PP1C, but with the C-terminal 72 residues of the M110 m subunit, a region which is 43 % identical to residues 87–161 of the M21 subunit. A fragment of the M21 subunit, M21-(M1–L146), which lacks the C-terminal leucine zipper, also bound to the M110 subunit, but two other fragments M21-(M1–E110) and M21-(E110–K186) did not. The M110 and M21 subunits were both found to be myosin-binding proteins. The C-terminal 291 residues of the M110 subunit, but not the C-terminal 72 residues, bound to myosin, but the N-terminal fragments M110-(M1—E309) and M110-(M1—S477) did not. Thus, the region of the M110 subunit that stimulates the dephosphorylation of myosin by PP1C is distinct from the region that targets PP1M to myosin. Remarkably, each myosin dimer was capable of binding about 20 tool M21 subunit and many of the M21-binding sites were located in the myosin rod domain. The potential significance of this observation is discussed. [ABSTRACT FROM AUTHOR]

Syringopeptin 25-A is a phytotoxic amphiphilic lipodepsipeptide containing 25 amino acid residues, produced by some isolates of the plant pathogenic bacterium Pseudomonas syringae pv. syringae. Previous papers have reported its covalent structure and some of its biological properties. Attention has now been directed to define its conformation in solution, a structural feature regarded as important for understanding its possible role in the bacterial colonization of host plants, and its toxic action on the plant cell. Here we report the stereochemistry of its amino acid components, the complete interpretation of the two-dimensional NMR spectra and NOE data, and finally the structure obtained by computer simulations applying distance geometry and molecular dynamics procedures. The confirmation of the syringopeptin 25-A in aqueous solution includes three different structural regions interrupted by rigid 2,3-dehydro-aminobutyric acid residues: a loop from residue 2 to 6, a helicoidal zone from 8 to 15, and the lactone ring from 18 to 25. The three dimentional structure of the lactone moiety is very similar to that of two previously studied bioactive lipodepsinonapeptides. Preliminary circular dichroism evidence of conformational variations in solution of trifluoroethanol, which stimulates a membrane-like environment, are also reported. [ABSTRACT FROM AUTHOR]

Biosynthetically directed fractional 13C labeling of proteinogenic amino acids is achieved by expression of proteins on a minimal medium which contains a mixture of [13C6]glucose and glucose with natural isotope abundance as the sole carbon source. Subsequent hydrolysis of the proteins yields the free amino acids. The observation of 13C-13C spin-spin scalar coupling fine structures in sensitive two-dimensional heteronuclear [13C,1H]-correlation spectroscopy (2D [13C,1H]-COSY) allows one to identify non-random 13C-labeling patterns arising from the incorporation of intact two-carbon and three-carbon fragments from a single source molecule of glucose into the amino acids. Since 2D [13C,1H]-COSY suffices to resolve all relevant resonances, the mixture of amino acids can be analyzed without further separation of its components. Probabilistic equations relate the observed multiplet intensities of the 13C fine structures to the relative abundance of the intact carbon fragments. They enable a quantitative analysis of the carbon flux in the network of biosynthetic pathways, thus using the proteinogenic amino acids as probes to study intermediary metabolism. This paper shows that biosynthetically directed fractional 13C labeling of amino acids provides an efficient analytical tool to quantitatively investigate glycolysis, pyruvate metabolism, pentose phosphate pathway, tricarboxylic acid cycle and C1 metabolism. Possible applications of the method include both the exploration of unknown biosynthetic pathways and the rapid elucidation of the response of a known biosynthetic reaction network to changes in growth conditions or genetic manipulations. [ABSTRACT FROM AUTHOR]

The subcellular distribution of hepatic alanine:glyoxylate aminotransferase 1 (AGT) has changed, under the influence of dietary selection pressure, on several o occasions during the evolution of mammals. In some species (e.g. human and rabbit) AGT is entirely peroxisomal; in other species (e.g. marmoset and rat) this enzyme is found in similar amounts in peroxisomes and mitochondria; in yet other species (e.g. cat) it is mainly mitochondrial. The molecular basis of the species-specific dual intracellular targeting of AGT has been partially elucidated in the human and rabbit (as examples of the first group), and in the rat and marmoset (as examples of the second group). As part of a wider study on the molecular evolution of AGT intracellular targeting, we report in the present paper the results of an investigation into the molecular basis of the subcellular distribution of AGT in the cat (as an example of the third group). Cat liver AGT cDNA has been cloned and sequenced, and shown to have a high degree of similarity to AGT from human, rabbit, marmoset and rat. Southern-blotting analysis showed that AGT in the cat is probably encoded by a single gene, as it is in other species. Transcript analysis by RNase protection indicated that almost all of the AGT mRNA would possess an open reading frame encoding a polypeptide of 414 amino acids and a molecular mass of 45,508 Da. The N-terminal 22 amino acids comprised the putative mitochondrial-targeting sequence (by analogy with the equivalent sequence in marmoset and rat pre-mitochondrial AGT). The very low level of peroxisomal AGT in cat liver is compatible with the absence of any RNase-protected transcripts initiating downstream of the first putative translation initiation codon (i.e. absence of any transcripts in which the mitochondrial-targeting sequence is excluded from the open reading frame). [ABSTRACT FROM AUTHOR]

Preparations of bovine lung surfactant D (SP-D) and conglutinin were examined by electron microscopy, gel-filtration and SDS/PAGE. SP-D is composed of non-covalently linked subunits, of 160 kDa, which each contain three, disulphide-linked, 44-kDa polypeptide chains. In the electron microscope a single 160-kDa subunit of SP-D appears as a 45.8 ± 3-nm-long rod connected to a small globular ‘head’. Particles were also seen which correspond to non-covalently linked dimmers, trimers and tetramers of the 160-kDa monomer subunit of SP-D. The tetramer structure contains 12 polypeptide chains and is very similar to the electron microscopy images and model reported by Strang et al. [Strang, C. J., Slayter, US., Lachmann, P. J. and Davis, A. E. (1986) Biochem. J. 236, 3811–389] for bovine conglutinin in which four 160-kDa subunits are disulphide-linked to give a molecule of expected molecular mass of 528 kDa. This study confirmed the findings by Strang et al. in the above paper for intact conglutinin and also emphasized that the rod-like structures, of length 37.6 ± 3.7 nm. Seen in the conglutinin subunits were significantly shorter than those in SP-D despite the close similarity in amino acid sequence (79% identity) and chain length between the two proteins. In addition, a truncated form of conglutinin was found in the conglutinin preparations, due to limited proteolysis of the Arg-Ala bond at position 54 in the 44-kDa chains. These truncated conglutinin chains yield a subunit composed of three shortened, non-disulophide-linked, chains and this subunit appears as a monomer with a rod length of 34.2 ± 2.8 nm in the electron microscope. On gel-filtration, a proportion of the SP-D preparation behaved, as expected, as a molecule with an apparent molecular mass of 600 kDa. The remainder of the SP-D preparation behaved as aggregated material with a molecular mass greater than 900 kDa which yielded no distinct structures in the electron microscope. Intact conglutinin was wluted at a position greater than 900 kDa but yet provided clear electron microscopy images of the tetramer structure described above. Although it is difficult to explain fully the anomalous behaviour of SP-D and conglutinin on gel-filtration, it is proposed that the natural form of both Sp-D and conglutinin is the highest distinct oligomer form seen in the electron microscope, i.e. a tetramer of the 160-kDa subunits in which the four rod-like elements are linked in a tail-to-tail fashion to a central core. [ABSTRACT FROM AUTHOR]

A 64-kDa membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell/cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth-phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysyl-endopeptidase-digested peptides of gp64 were determined by protein analysis; all six asparagine- linked glycosylation sites (Asn-Xaa-Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins. [ABSTRACT FROM AUTHOR]

A protein is usually classified into one of the following four structural classes: all α, all β, (a + B) and α/β. In this paper, based on the maximum correlation-coefficient principle, a new formulation is proposed for predicting the structural class of a protein according to its amino acid composition. Calculations have been made for a development set of proteins from which the amino acid compositions for the standard structural classes were derived, and an independent set of proteins which are outside the development set. The former can test the self consistency of a method and the latter can test its extrapolating effectiveness. In both cases, the results showed that the new method gave a considerably higher rate of correct prediction than any of the previous methods, implying that a significant improvement has been achieved by implementing the maximum-correlation-coefficient principle in the new method. [ABSTRACT FROM AUTHOR]

Analyses of unimolecular electron self-exchange reactions provide a comparatively simple and direct approach to understanding biological electron transfer. Such studies are currently limited by a lack of well characterised aggregating systems. In the presence of sodium hexametaphosphate, cytochrome c forms stable protein aggregates as a result of binding hexametaphosphate at a single site on its surface (preceding paper in this issue of the journal). Here we report the location of the principal polyphosphate binding site on the surface of cytochrome c for both hexametaphosphate and a second polyphosphate, tripolyphosphate determined using 1H-NMR spectroscopy in conjunction with the relaxation probe potassium hexacyanochromium(III). Addition of either hexametaphosphate or tripolyphosphate to ferricytochrome c in the presence of the relaxation probe causes a decrease in intensity of several resonances in the paramagnetic difference spectrum, including Phe82 ortho/meta, I1e85 δ methyl and Ile9 γ methyl. Together these effects put the site of polyphosphate binding close to lysines 13, 86, and 87. Additionally the effect of sodium tripolyphosphate and sodium trimetaphosphate on cytochrome c aggregation is described. The potential role of this site in anion-induced cytochrome c aggregation is discussed. [ABSTRACT FROM AUTHOR]

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60-kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild-type Rous sarcoma virus (SRD-FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v-src, the mAb327 [Lipsich. L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352–360] and to be phosphorylated in vitro at serinc/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c-src protein immunoprecipitated with mAb327 from extracts of NIH c-src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor-bearing rabbit (TBR) serum and reported in an earlier work [David-Pfeuty, T. & Hovanessian, A. (1984) Eur. d. Biochem. 140, 325-342], together with those reported here, suggest that the TBR-immunoprecipitated pp60v-src coprecipitates with a cellular protein related to the 60-kDa subunit of the Ca2+ / calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v-src, but not of pp60c-src, are shown to contain a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates a 52-kDa protein substrate. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in secretory proteins and in various classes of cellular proteins has been quantified and compared to protein-bound carbohydrate sulfate. 2. HepG2 cells and fibroblasts, two cell types showing only the constitutive pathway of secretion, and PC12 cells, which show both the constitutive and the regulated pathway of secretion, were subjected to pulse-chase and/ or long-term labelling with [35S]sulfate and [³H]tyrosine, followed by analysis of proteins in the cells and medium. Under both conditions of labelling, 65-92% of the protein-bound tyrosine sulfate and 44-84% of the protein bound carbohydrate sulfate were found to be secretory. In HepG2 cells, the frequency of sulfation of tyrosine residues, which can be determined independently from protein abundance and the rate of protein synthesis, was 8-22 times higher in proteins secreted into the medium than in cellular proteins. 3. All cell lines studied contained significant amounts, not only of carbohydrate sulfate, but also of tyrosine sulfate in specific cellular proteins. As shown for fibroblasts, these tyrosine-sulfated proteins were retained within the cells for at least 100 min of chase following a pulse with [35S]sulfate and were almost completely recovered in a light membrane fraction after subcellular fractionation. 4. Lysosomes were found to contain small, but significant, amounts of protein-bound tyrosine sulfate in addition to protein-bound carbohydrate sulfate. Protein-bound tyrosine sulfate in lysosomes reached a peak at 20 min of chase and rapidly disappeared thereafter, whereas protein-bound carbohydrate sulfate accumulated after 20 min of chase. Examination of the known sequences of eleven lysosomal enzymes revealed the presence of potential tyrosine sulfation sites in five of them. 5. Our results show that secretory proteins are the most abundant, but not exclusive, in vivo substrates for tyrosine sulfation and suggest the presence of soluble tyrosine-sulfated proteins in lysosomes and other, as yet unidentified, organelles of the secretory pathway. In the following paper in this journal we describe the abundance of tyrosine sulfate in integral membrane proteins. [ABSTRACT FROM AUTHOR]

1. The abundance of tyrosine sulfate in membrane proteins was quantified in four different cell lines and compared to that in soluble cellular and secreted proteins. 2. Upon metabolic labelling of HepG2, Ltk-, AtT20 and PC12 cells with [35S]sulfate or [³H]tyrosine, a fraction enriched in integral membrane proteins was found to contain small, but significant, amounts of protein-bound tyrosine sulfate (up to 2.5% of the total cellular plus secreted protein-bound tyrosine sulfate). On the other hand, the frequency of sulfation of tyrosine residues of membrane proteins was within the same order of magnitude as that of secreted proteins, indicating that the low abundance of tyrosine sulfate in membrane proteins was largely a reflection of the low abundance of these proteins themselves. Consistent with this conclusion were the results of an analysis showing that 14 out of 32 selected membrane-spanning proteins contain potential tyrosine sulfation sites. 3. In HepG2 cells, three tyrosine-sulfated integral membrane glycoproteins of molecular mass 100, 125 and 150 kDa were identified. Characterization of the 150-kDa tyrosine-sulfated membrane protein revealed that it was protected from proteolysis in intact cells, suggesting a localization in an intracellular organelle. 4. Together with the results reported in the preceding paper in this journal, our data suggest that tyrosine sulfation occurs in various classes of trans-Golgi-derived proteins, soluble as well as membrane, and extracellularly exposed as well as intracellularly retained, proteins. This suggests that tyrosine sulfation may have a variety of physiological functions, depending on the individual tyrosine-sulfated protein or protein class. [ABSTRACT FROM AUTHOR]

Transglutaminases (EC 2.3.2.13) catalyze the formation of ε-(γ-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma;-carboxamide groups of protein-bound glutamine residues. These enzymes arc involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a eDNA of guinea-pig liver transglutaminase between the Ncol and Pstl sites of an expression vector, pKK233-2, and produced the Liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyt-terminal sequences. However, the α-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acctylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km, values for substrates in the histamine incorporation into acetyl α1¹-casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases. These results indicated that the Nα-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme. [ABSTRACT FROM AUTHOR]

Two types of linkages between the carbohydrate and the peptide moiety in the glycopeptide from Ascobolus furfuraceus are described. Treatment with mild alkali produced β-elimination of a small oligosaccharide. Evidence for the O-glycosidic linkage was provided by (a) increase in absorbance at 240 nm, (b) decrease in threonine and serine content after the alkaline treatment and (c) detection of tritiated oligosaccharide following alkaline NaB³H4 reduction. Mannose is the sugar involved in the O-glycosidic linkage. The remaining glycopeptide was branched by galactofuranose units, which were selectivity released by mild acid hydrolysis. The N-glycosidic linkage of the sugar chain was conclusively proved by cleavage with endo-β-N-acetyl-glucosaminidase. Sequential NaB³H4 reduction and acid hydrolysis gave [³H]glucosaminitol. The structure of the sugar chain was studied by 13C NMR spectroscopy and by methylation analysis. [ABSTRACT FROM AUTHOR]

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the sucking mouse assay respectively. [ABSTRACT FROM AUTHOR]

The identities of the protein phosphatases involved in the regulation of hepatic glycolysis, gluconeogenesis and aromatic amino acid breakdown were investigated using 6-phosphofructo-1-kinase, fructose-1,6-biphosphatase, L-pyruvate kinase, phenylalanine hydroxylase and the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates. Purified preparations of protein phosphatases-1, 2A, 2B and 2C exhibited activity towards all five substrates in vitro, although phosphatases-1 and 2B were only weakly active. Studies in liver extracts using inhibitor-2 and trifluoperazine, which inhibit protein phosphatase-1 and 2B, respectively, confirmed that these phosphatases are unlikely to be important in dephosphorylating these substrates in vivo. Sequential fractionation of rat liver extracts by anion-exchange chromatography and gel-filtration failed to resolve any protein phosphatases acting on each substrate, apart from protein phosphatases-2A and 2C. The present results, together with those described in the following paper (in this journal) indicate that under the assay conditions used, protein phosphatase-2A is the most powerful phosphatase acting on each substrate, although protein phosphatase-2C contributes a significant percentage of the activity towards 6-phophofructor-1-kinase. No clear evidence was obtained for a role of metabolites in the regulation of dephosphorylation of the five substrates. This study reinforces our contention that only a few serine-specific and threonine-specific protein phosphatase catalytic subunits participate in cellular regulation. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. a phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the chage in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary hitory, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs. [ABSTRACT FROM AUTHOR]

According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side-chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made. [ABSTRACT FROM AUTHOR]

The partial ammo acid sequence of dihydrofolate reductase (DHFR. EC 1.5,1.3) from human KB/6b cells hi been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradation and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequence. A comparison of the proposed human DHFR sequence with the previously known sequent of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Owls and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704. 163- 173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight micrasornal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine- 15 by arginine. This paper brings the final proof that two iso-cytochromes b5- exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed. [ABSTRACT FROM AUTHOR]

Peptide sequences obtained from hen ovotransferrin are compared with the complete amino acid sequence of the protein deduced from a cDNA sequence (Jeltsch and Chambon, preceding paper). Of the 705 positions of the whole protein 605 can be matched by the peptide sequences. Some possible discrepancies between the two methods are pointed out. The two halves of the chain show marked similarities in their sequences with 37 % identical residues. The positions of the 15 disulphide bridges are shown; there are 6 homologous bridges in each half of the molecule and 3 extra bridges which occur only in the C-terminal half. The terminal residues of the half- molecule fragments obtained by limited proteolysis are identified. The two domains are joined by a 9-residue connecting peptide. Sequence variability has been found at 9 positions. The sequence of hen ovotransfernn is compared with the partial sequence available for human transferrin. From this some tentative conclusions about the identities of the metal-binding residues and about the evolution of transferrin are reached. [ABSTRACT FROM AUTHOR]

The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species. Bovine gastricsin was prepared from commercial extracts of adult bovine veils by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Us molecular weight was estimated by gel filtration to be 32800. Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pi values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A—Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A—Sepharose 4B. Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins. Bovine gastricsin which is inactivated by reaction with diazoacetyl-DL-norleucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with χ-casein as substrate is only half of that of chymosin and pepsin A. [ABSTRACT FROM AUTHOR]

The light chain fraction was separated from chicken gizzard muscle myosin. After S-carboxymethylation or performic acid oxidation, two light chain components (20 000-M[subr] and 17 000-M[subr] chains) were isolated by chromatography on a column of DEAE-cellulose in the presence of 4 M urea. Tryptic peptides of the S-carboxymethylated 20 000-M[subr] chain were isolated, and their sequences were determined. The alignment of these tryptic peptides in the chain was deduced from the amino acid compositions and from the partial sequences of peptic peptides of the oxidized protein. The established sequence consists of 171 amino acids and its calculated molecular weight is 19692. Comparing the sequence with those of L-2 chains from chicken and rabbit skeletal muscle myosins, 81 and 78 amino acid substitutions were recognized, respectively, including insertions and/or deletions. [ABSTRACT FROM AUTHOR]

The brain specific S-100 protein is a mixture of two components S-100a and S-100 b with a subunit composition αβ or β2 respectively. The amino acid sequence of the β subunit has been previously determined. This paper presents the sequence of the α subunit in the S-100a protein. The α subunit consists of 93 amino-acid residues and has a relative molecular mass of 10400. The sequence shows extensive homology (58 %) with that of the β-subunit and shares an apparent calcium binding site in the C-terminal half of the molecule, suggesting a close evolutionary relationship between these subunits. [ABSTRACT FROM AUTHOR]

Describes an application of two-dimensional, nuclear magnetic resonance techniques for the study of proteins. Amino acid residue; Molecular weight; Glycyl residue spin systems; Spin-spin coupling constants; Protein conformation.

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]

We have developed a semi-empirical method to determine the subunit molecular weight of a protein. The method is a minor modification of the Edman degradation and is based on a simple chemical procedure to modify specifically the N-terminal NH2 group. Initially, all NH2 groups, including the ε-NH2 groups of lysine residues and the N-terminal α-NH2 group, are reacted with phenylisothiocyanate and the protein derivative is subjected to one step of the Edman degradation. The newly exposed N terminus is then reacted with radioactively labelled phenylisothiocyanate. A value for the subunit molecular weight can be obtained from an analysis of the incorporated radioactivity and the amount of the protein in the sample. The molecular weight of five different proteins have been determined by this method. Our method is particularly useful for proteins containing lipid or sugar components and also for relatively small peptides. The procedure described in this paper for the specific modification of the N terminus has been found to be a powerful tool for protein sequencing. [ABSTRACT FROM AUTHOR]

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

Sheep-serum transferrin shows marked polymorphism and more than 20 alleles have been identified although only 4 or 5 of these have a frequency higher than 1%. Each of the alleles has two bands in starch-gel electrophoresis, corresponding to a major and a minor fraction. This paper describes the isolation and partial characterisation of the two fractions from the transferrin of sheep homozygous for Tf B. The purification consisted of: (a) precipitation by ammonium sulphate, (b) chromatography on CM-cellulose and, finally (c) chromatography on DEAE-Sephadex. The purification procedure had no effect on the electrophoretic mobility of the two fractions and they appeared homogeneous by starch-gel and polyamide-gel electrophoresis and by immuno-electrophoresis. The amino-acid compositions of both fractions were very similar and the sequences of the first eight amino-acid residues: Ser-Pro-Glu-Lys-Thr-Val-Arg-Trp-were identical for both bands. These results, and the fact that the two fractions are found in all genetic variants and always have the same relative mobility strongly suggest that the differences do not lie in the polypeptide chain. From the results of hydrolysis by neuraminidase and assay of sialic acid, the major and minor fractions most probably contain two and three sialic acid residues (exclusively N-acetylneuraminic acid) respectively, thus explaining the different electrophoretic mobilities. The sialic-acid content has been calculated on the basis of a molecular weight of 77500 as determined both by low-speed equilibrium ultracentrifugation and by Archibald's method. The following physico-chemical constants have been established: absorption coefficient at 280nm, A1 mg/cm³1 cm = 1.25 ± 0.01 cm² · mg-1; partial specific volume, &vmacr; = 0.734 cm³ · g-1; sedimentation coefficient s20.wo = 5.15 S; refractive-index increment, dn/dc = 0.173 cm³ · g-1. [ABSTRACT FROM AUTHOR]

A new and efficient method for preparation of pure phosphoglyceromutase from baker's yeast (Saccharomyces cerevisiae) is described. Proteolytic alterations of the enzyme during extraction can be minimized by grinding the dried yeast with aluminium oxide at low temperature. Yeast phosphoglyceromutase contains four highly similar, probably identical subunits of molecular weight 28000, a conclusion based on the following observations. Polyacrylamide gel electrophoresis containing dodecylsulphate or urea gives a single band, indicating that the enzyme is composed of four subunits similar in their molecular weight and net charge. Cyanogen bromide cleavage and tryptic digestion of the enzyme yield the number of peptides expected for identical subunits from the amino acid composition analysis. The purified phosphoglyceromutase preparation has bisphosphoglyceromutase activity synthesizing 2,3-bisphosphoglycerate from 1,3-bisphosphoglycerate and 3-phosphoglycerate. It has been reported that yeast phosphoglyceromutase catalyzes the hydrolysis of 2,3-bisphosphoglycerate at the same active site which catalyzes the phosphoglyceromutase reaction [Sasaki, R. et al. (1971) Biochim. Biophys. Acta, 227, 584–594, 595–607]. Immunological studies and chemical modification experiments indicate that bisphosphoglyceromutase activity also is due to the phosphoglyceromutase protein and involves amino groups which have been shown to be essential for the other two activities. [ABSTRACT FROM AUTHOR]

The primary structure of protein S8 from the 30-S ribosomal subunit of Escherichia coil was determined mainly by automatic Edman degradation using a modified Beckman protein sequenator and the solid-phase sequenator of Laursen. The complete sequence, containing 109 amino acids, was derived by analysing peptides from tryptic, chymotryptic, thermolysin, staphylococcal protease and cyanogen bromide digestion of the protein. The amino acid composition was found to be (aspartic acid)6, (asparagine)3, (threonine)5, (serine)5, (glutamic acid)7, (glutamine)6, (proline)5, (glycine)6, (alanine)11, (valine)9, (methionine)4, (isoleucine)7, (leucine)9, (tyrosine)3, (phenylalanine)3, (lysine)11, (arginine)8, (cysteine)1. S8 is a basic protein and binds to the 16-S RNA; knowledge of its sequence is necessary for a detailed study of its interaction with the ribosomal RNA. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the light chain of the mouse monoclonal MOPC 173 immunoglobulin molecule (IgG2a,χ) is presented. This kappa chain contains 214 residues. Comparisons of this sequence with murine kappa chains already published by other workers bring a confirmation of the large size of the murine Vχ chain pool. A complete identity was found with the constant region of the light chain of MOPC 21 from residue 97 to residue 214. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of CNBr fragment H4 of the murine immunoglobulin MOPC 173 (IgG2a,χ) has been determined, thus completing the sequence determination of the entire heavy chain. The H4 fragment contains 150 residues, and extends from residue 105 to residue 254 of the heavy chain, which appears thus to be composed of 447 amino acids residues. This fragment contains the end of the V region, the switch peptide, the CH1 domain, the hinge region and the beginning of CH2. Sequence comparisons suggest that the CH1 domain is highly conserved in evolution, and allows the definition of two additional isotypic-specific regions. [ABSTRACT FROM AUTHOR]

The apamin sequence has been synthesized by the solid-phase procedure. The synthetic peptide showed the same physicochemical and chemical properties as natural apamin in the following tests: paper electrophoresis, amino acid analyses after acid and enzymatic hydrolyses, sequence analysis, electrophoreses after tryptic and chymotryptic digestions. Synthetic apamin was as active as natural apamin in the neurotoxic assay in mice (LD50, after subcutaneous injection, for the 20-g mouse: 58 μg). [ABSTRACT FROM AUTHOR]

By conversion of prochymosin into active chymosin an N-terminal segment of 42 amino acid residues is liberated. In one activation experiment this segment was recovered in two peptides; in a second experiment the activation segment was cleaved into three peptides. The primary structures of the peptides have been determined. Overlaps between these peptides and between the activation segment and the active enzyme have been obtained from peptides produced by tryptic digestion of denatured prochymosin. Comparison of the amino acid sequences of the activation segments from bovine prochymosin, bovine pepsinogen and porcine pepsinogen shows considerable homology. [ABSTRACT FROM AUTHOR]

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of α-H atoms of the amino acid substrate with 3H2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The α-H of the accumulating j8-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled α-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic β-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of β-H atoms in the amino substrate or of 18O-labelled β-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse α-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonine, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, Ki, is equal to the Michaelis constant, Km, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, α-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic α-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific β-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of α,β-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing β-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper. [ABSTRACT FROM AUTHOR]

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesonteroides reversibly which Ki = 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+ Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme- inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by I -fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ and glucose 6-phosphate protect against inhibition by 1-fluoro-2,4-dinitrobenzene. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically. [ABSTRACT FROM AUTHOR]

The sequences of the 119 amino acids of bovine and porcine luteinizing hormone β-subunits were determined, 17 amino acid replacements being observed between these species. Variability in the primary structure was observed for porcine β-subunit, in that position 10 is occupied by both arginyl and glutamyl residues. No free amino terminal residue was detected for both porcine and bovine β-polypeptide chains and both exhibited carboxy-terminal heterogeneity. The homology of the β-subunits of luteinizing hormone from various species and the β-subunit of the bovine thyroid-stimulating hormone is discussed as the subunit of both hormones can combine with a similar α-subunit. [ABSTRACT FROM AUTHOR]

The primary structure of porcine luteinizing hormone α-subunit was established by determination of the amino acid sequence of its tryptic peptides, which were aligned by homology with the known sequence of ovine luteinizing hormone α-subunit. Its polypeptide chain is shorter by six amino acid residues at its amino-terminus, when compared to the ovine α-subunit. Furthermore four amino acid replacements are observed in the porcine as compared with the ovine chain. Heterogeneity of both carbohydrate prosthetic groups of porcine luteinizing hormone α-subunit is indicated by the different sugar compositions of the glycopeptides isolated in the course of this work. [ABSTRACT FROM AUTHOR]

The biosynthesis of melittin, the main toxin of bee venom, was studied in vivo by feeding radioactive amino acids to honey bees. Extracts from venom glands were analyzed for the presence of labeled melittin and other components. Radioactivity was first incorporated into another peptide which is considered to be a precursor of melittin. This conclusion is based on the observed labeling kinetics demonstrating the transient formation of this peptide. Furthermore, the structural similarity between melittin and this component could be established. Evidence is presented showing that it differs from melittin at the amino end. The precursor peptide could not be detected in the ejected venom. [ABSTRACT FROM AUTHOR]

Our previous studies [1,2] on maleyl bovine αS1-B casein were concerned with the isolation and amino acid composition of the tryptic peptides designated as Tm peptides. The sequence of the COOH-terminal Tm peptide containing 48 residues was also reported. In the present study, we report the overlaps of all the cyanogen bromide peptides as well as the Tm peptides from αs1-B casein. The αs1-B casein which contains five methionyl residues was cleaved with cyanogen bromide. The six expected peptides obtained by this treatment and designated as CN peptides, were separated initially on Dowex-50X2 and further purified by Sephadex column chromatography. The amino acid composition of these six purified CN peptides was determined. The results were in perfect agreement with the amino acid composition of αs1-B casein deduced from the analysis of Tm peptides. The three Tm peptides from αs1-B casek@ which contained methionyl residues were also cleaved by cyanogen bromide. The fragments obtained were purified by either paper chromatography or Sephadex column chromatography and the amino acid composition of these purified peptides was determined. Of the eight peptides thus obtained, three were found identical with the CN peptides obtained directly by BrCN treatment of the αs1-B ease@. The remaining three CN peptides from αs1-B casein for which identical fragments could not be located fun the Tm peptides, were digested with trypsin (in two eases after maleylation) and the resulting fragments were purified by Sephadex column chromatography. These trifle peptides provided the remaining gaps. This permitted in to locate the relative position of all the CN peptides in the αs1-B casein molecule. These results also indicate the location of all the Tm peptides except 2 in the αs1-B casein molecule. Further, the chymotryptic peptides from one of the CN fragments of αs1-B casein provided the missing overlaps. [ABSTRACT FROM AUTHOR]

In order to determine the primary structure of the bovine β-caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr). The tryptic hydrolyzate was separated by column chromatography on Dowex-50. After additional purifications, 13 peptides numbered Ti to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino-acid analyses were carried out on all these peptides. Peptide T1 contain 2 Arginyl residues, one of which is NH2-terminal. This peptide represents the NH2-terminal end of the β-casein. Its composition is identical to that reposed by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β-casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2terminal. The COOH-terminal peptide was identified with peptide T4, devoid of basic aminoacids. The 14 tryptic peptides account for all the residues of the β-casein A2 when compared with the already published amino-acid analyses of the whole protein [2, 3]. Several other peptides, originating from incomplete or non-specific cleavages, were also obtained. Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β-casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G-50. The seventh peptide was obtained by chromatography on Dowex-50 of the whole CNBr-digest. After additional purifications, the ammo-acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2terminal end of β-casein. The COOH-terminal end of β-casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr-peptides account for all of the amino-acid residues of β-casein A2. β-casein A2 has 208 amino-acid residues. Its molecular weight is very close to 24 000. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

The complete sequence (153 amino acids) of horse heart myoglobin has been established. The sequence of a peptide isolated from chymotrypsin hydrolyzates of globin allowed to fill a gap in the tentative sequence previously proposed; this peptide was studied by means of pepsin hydrolysis, action of N-bromosuccinimide on histidyl bonds, and Edman degradation method associated with dansylation. After hydrolysis of globin by chymotrypsin treated by TLCK, the same major peptides were found as after hydrolysis by non-treated chymotrypsin. These peptides were identified by ionexchange resins and bidimensional paper chromatography, and also by the determination of their amino-acid composition. The suppression of secondary cuts explains the presence of new peptides, whose the composition and N-terminal Sequences were determined, it was then possible to confirm several sequences which could be previously deduced from the study of split products after cyanogen bromide treatment. The specificity of chymotrypsin towards certain types of peptide bonds is discussed. Horse myoglobin, as compared with sperm whale myoglobin, contains 18 differences: 17 substitutions and one inversion. The major part of the substitutions are located ih N- and C-terminal sequences: they are all punctiform, resulting from the replacement of only one base in each codon. [ABSTRACT FROM AUTHOR]

The behavior of N-acetylphenylalanyl-tRNA as an initiator of phenylalanine polymerisation in an Escherichia coli cell-free system shows some analogy with the behavior of formylmethionyl- tRNA during the synthesis of natural proteins. At low Mg++ concentrations, polyphenylalanine chain initiation depends on N-acetylphenylalanyl-tRNA and the same initiation factors which are required for protein chain initiation with formylmethionyl-tRNA. However in a rabbit reticulocyte cell-free system, N-acetylphenylalanine is not incorporated into polyphénylalanine at any Mg++ concentration. In this paper, we showed that : a) N-acetylphenylalanyl-tRNA did bind to reticulocyte ribosomes in the presence of poly U. This binding was GTP dependent and needed a factor which was extracted by washing ribosomes with salt (NH4CI 1 M); b) the bound N-acetylphenylalanyl-tRNA was partially released by puromycin or by addition of phenylalanyl-tRNA. In this last case, incorporation of N-acetylphenylalanine into polyphenyl- alanine was noticed (ratio N-acetylphenylalanine/phenylalanine = 1/20); c) the ribosomal wash fluid also greatly enhanced phenylalanyl-tRNA binding to salt- treated ribosomes, in the presence of poly U and GTP. The maximum binding was obtained with the same concentrations of Mg++ and K+ as for N-acetylphenylalanyl-tRNA binding. It was not possible to separate on a DEAE-cellulose column the factors that were required for phenyl- alanyl-tRNA and acetylphenylalanyl-tRNA binding. On the other hand, a preparation of the binding enzyme from reticulocyte supernatant enhanced both phenylalanyl-tRNA and acetyl- phenylalanyl-tRNA binding to ribosomes; d) the optimum Mg+÷ concentration for phenylalanine polymerisation was higher with salt- washed ribosomes than with sucrose-washed ribosomes. Adding the wash fluid shifted the Mg++ optimum back to the lower value. N-acetylphenylalanyl-tRNA was not required for this shift as noticed in Escherichia coli. One likely explanation of our results with reticulocytes is the possibility that the ‘binding enzyme’ of Schweet et al., which was present in our ribosome wash fluid, could work with both N-acetyl as well as with phenylalanyl-tRNA. Thus, the inability to incorporate N-acetylphenyl- alanine might be due to a competition between these two compounds for the same enzyme and the same ribosomal site. If this is indeed the case, it is in marked contrast to results obtained with Escherichia coti and could reflect a basic difference in the mechanism of protein chain initiation. [ABSTRACT FROM AUTHOR]

Phylogenic research on the haptoglobin of different animal species led us to study the structure of rabbit haptoglobin. Rabbit haptoglobin, whose homogeneity was checked according to several criteria, was submitted to hydrolysis by pronase (72 h, 39°, pH 8.2); the hydrolysate was then filtered through Sephadex G-25, and the resulting glycopeptide fraction was separated on Dowex 50-X2 (H+) into two parts, one (A) not retained by the resin, the other one (B) collected after elution with 0.5 M NaCl. Amino-acid and carbohydrate composition, nature of end groups, and timing of sialic acid release, were determined on fraction A; from the amount of aspartic acid, the molecular weight was estimated to be no less than 2860. Fraction B, which amounted to one third of fraction A, could not be analysed on account of the insufficient quantity available. Quantitative paper electrophoresis at pH 3.5 and 6.2 yielded 8 glycopeptides from fraction A and 2 from fraction B. These various glycopeptides showed notable differences in their amino-acid composition, mainly with respect to glutamic acid, proline, glycine and histidine. On the contrary, no significant differences were found in the carbohydrate composition, with the exception of the oses N-acetylneuraminic acid ratio, which varies from 2 to 10. These results suggest 6 oligosaccharide units to be present in rabbit haptoglobin, as compared to 7 in human haptoglobin, bound to peptidic fractions varying in their amino-acid composition in the neighbourhood of the carbohydrate-peptide linkage. Although the precise nature of this bond has not been ascertained, it does not appear to be an O-glycosidic one. [ABSTRACT FROM AUTHOR]