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Viene descritto un metodo rapido e sensibile per la rilevazione delle dipeptidasi su carta.

1. The gut-contracting peptide components Fa, Fb and Fc were prepared from crude substance P preparations made from cortical and subcortical tissues of human and bovine brain. 2. Fc preparations of human cortex, amygdala, hypothalamus, and red nucleus were then chromatographed on columns of DEAE-cellulose. 3. These experiments and such using paper chromatography, thin-layer chromatography, and paper electrophoresis led to results which point to the possible existence in brain of numerous polypeptides with pharmacological, i.e. gut-contracting activity. 4. There exist species differences between active peptide fractions of corresponding areas of human and bovine brain.

Synthetic substance P (SP) was compared with the gut-contracting peptide Fa from bovine subcortex by means of thin-layer chromatography, paper electrophoresis, enzymatic digestion, and biological assays. With all four techniques differences in behaviour between Fa and synthetic SP were found, which lead to the conclusion that both active principles are similar but not identical. Both materials could also be distinguished from synthetic physalaemin with the aid of paper electrophoresis and thin-layer chromatography.

We have been unable to confirm the recent report that kaolinite catalyzes the polymerization of aqueousd- andl-aspartic acid at different rates. In experiments wheredl-Asp was used, no induced optical rotation was found in the reaction solution. No evidence for polymer (or other product) formation was found whenl-Asp-2-14C was used, and products were searched by paper chromatography and X-ray film autoradiography. Asp is adsorbed by kaolinite, but no selectivity for one or the other enantiomer was observed.

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel nitration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and nine minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor.

Determination of the primary structure of the peptidoglycan of 8 strains of Peptococcus showed that 4 different peptidoglycan types occur. P. prevotii ATCC 9321 and P. grigoroffii H40/10 contain the l-Lys-d-Glu type with glycine in position 1 of the peptide subunit. P. variabilis ATCC 14955 and H 39/5, P. activus H22/12 and P. anaerobius H/7 show the l-Lys-Gly type with glycine in position 1 of the peptide subunit. P. aerogenes ATCC 14963 contains the l-Orn-d-Glu type and P. saccharolyticus ATCC 14953 the l-Lys-Gly4, l-Ser1type. The occurrence of these different peptidoglycan types is a valuable criterion for the classification of peptococci. The following conclusions were made: P. saccharolyticus had to be excluded from Peptococcus. P. variabilis and P. anaerobius can be united within one species, whereas P. prevotii ATCC 9321 and P. aerogenes ATCC 14963 can not be included in one species as has been suggested before. P. grigoroffii H40/10 and P. activus H22/12 were wrongly classified.

Cleavage with cyanogen bromide was employed to investigate the N-terminal amino acid sequence of the λ-chains of normal porcine immunoglobulin. The N-terminal homoserine-containing nonapeptide was isolated from all fractions of the λ-chains in a yield of 60% of theory. Its amino acid sequence was determined by mass spectrometry and was found to be uniform. Other homoserine-containing fragments were obtained from the cyanogen bromide hydrolyzate of the λ-chains with split disulfide bonds. Partial amino acid sequence of these fragments provided evidence that methionine in position 9 is present in more than 60% of variants and obviously has an invariant character. Variants of the λ-chains exist (by estimate not more than 15% of the total λ-chains) which, in addition to the invariant methionine residue in position 9, possess a variable methionine residue in the section between positions 46 and 51. The data obtained indicate that the sequence of 23 amino acid residues from the N-terminus of porcine λ-chains is uniform with the exception of a single replacement (in position 13). Moreover, in comparison with the sequence of λ-chains of man, mouse and birds, the sequence displays several species-specific replacements and a deletion of threonine which occupies position 5 in the chains of the other species. In the pig, the λ-chains represent the predominant type of light chains (some 70%). Hence it follows that some 50% of the light chains in this species possess a uniform amino acid sequence in the N-terminal section. These facts are difficult to reconcile with the germline theories of genetic control so that mechanisms of somatic diversification of immunoglobulins must be envisaged.

The addition of mRNA preparations to a number of heterologous cell-free systems has been shown to result in the synthesis of immunoglobulin light chains. This paper reports the results of experiments in which an mRNA fraction prepared from MOPC 21 mouse myeloma cells has been used to stimulate the synthesis of immunoglobulin heavy chains in rabbit reticulocyte lysates. The synthesis of heavy chains in a homologous cell-free system containing either microsomes or membrane-derived polysomes is also described. The synthetic product has been characterised by finger-printing in each case. The results reveal the presence of a unique methionine-containing peptide in digests of the product of the rabbit reticulocyte cell-free system. This peptide is not observed in corresponding digests of heavy chains synthesized in vivo or in the homologous cell-free system in vitro.

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N-ethyl-[1-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential thiol group was isolated and found to contain: Leu-Gly-Tyr-Ile-Thr-[14C]Cys-Pro-Gly-Ser-Asn-Leu-Gly-Thr-Leu-Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle. In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ≃ 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.

At least three peptides accumulating in uraemic serum inhibit glucose utilization in the rat diaphragm. The nature of these peptides is unknown. Their amino acid composition was determined. This finding is the first direct evidence differentiating the pathogenesis of abnormal glucose utilization in uraemia from that in diabetes.

Cell-free protein synthesis has been studied in heterologous systems using rabbit reticulocyte postribosomal supernatant and ribosomes obtained from HeLa or mouse myeloma cells. These cell-free systems are quite active and incorporate labelled amino acids linearly for more than 30 min. The proteins synthesized have been analyzed by acrylamide gel electrophoresis, column chromatography and paper electrophoresis of the tryptic peptides. More than 70% of the protein synthesized with either type of ribosomes is α chain of rabbit hemoglobin; a small amount of β chain is also synthesized. This result can only be explained by the presence in the postribosomal supernatant of reticulocytes of messenger RNA for hemoglobin. This mRNA is translated more efficiently than the endogenous mRNA present on HeLa or myeloma ribosomes; previous measurements of the mRNA of reticulocyte postribosomal supernatant indicate that it is less on a weight basis than 1/20 of the mRNA associated with the corresponding amount of ribosomes used in an incubation. The preferential translation of mRNA present in the supernatant, presumably in a ribonucleoprotein particle, is discussed.

A technique is described for the detection of cystine or peptides containing cystine or its derivatives in digests of keratin. The technique involves the preparation of radioactive wool by the intravenous injection of experimental animals with (35S) cystine followed by the growth of keratinolytic fungi upon the labelled wool and the detection of soluble compounds in the wool digests which contain cystine or its derivatives by paper chromatography and autoradiography.

Proteolysis of horse heart cytochrome C with pepsin for 3 min produces a large heme peptide, which was shown to be a mixture of the sequences 1–64 and 1–66. By ion exchange chromatography, gel filtration and preparative paper chromatography the heme-free fraction was resolved into seven peptide fragments, corresponding to sequences 65–94, 65–96, 67–94, 67–96, 67–80, 95–104 and 97–104.

Following the procedure of Schramm for the synthesis of polynucleotides and polysaccharides, homopolymers ofdl-leucine,dl-phenylalanine,dl-serine, anddl-valine have been prepared in yields of 13 to 57 % through the mediation of a polymetaphosphate ester. Copolymers of the amino acids also have been prepared in lower yields (4–5 %). Infrared spectra show that the polymers are not diketopiperazines and that the polymers ofdl-leucine,dl-phenylalanine, anddl-valine are polypeptides. Conversions of as much as 57% and degrees of polymerization of approximately 12 were obtained for polyleucine. Small peptides containing possibly 2 to 3 leucine residues were detected and isolated as possible intermediates in the leucine polymerization reaction. For the polymerization ofdl-valine, a temperature of 60°C, a reaction time of 10–24 h, and a ratio of polymetaphosphate ester to amino acid of 3:1 appeared to give the best results. The Schramm procedure was initially suggested as a chemical evolution model for the formation of biological polymers under prebiotic conditions. Although the significance of this reaction to prebiological organic chemistry may be questioned, it still offers a mechanistic model for the study of the synthetic reactions involving polyphosphates which are indirectly relevant to abiotic molecular evolution and the problem of the origin of life.

Tryptic and chymotryptic digestion of peptide B, the N-terminal fragment obtained by cyanogen bromide cleavage of thioredoxin, provided the information required to determine the contiguity of the peptic fragments studied previously. The complete sequence of the 37 residues of peptide B could thus be established. Peptide B contains the active center of thioredoxin. Its amino acid sequence was found to be: . This sequence supports earlier conclusions drawn from fluorescence studies of thioredoxin, which indicated a close relationship between one or both tryptophan residues and the disulfide bridge of the active site. Peptide maps of the tryptic digests of carboxymethylated thioredoxin and of the two cyanogen bromide fragments are described. Amide nitrogen analyses of thioredoxin and the two cyanogen bromide fragments demonstrate that all seven amide groups of thioredoxin are localized in the C-terminal fragment (peptide A).

The peptide composition of variants of Sindbis virus which differ in plaque-type, elution pattern from calcium phosphate columns and virulence in mice was analyzed. Differences in peptide number or composition were not detected in these variants. Since the Sindbis virion has an outer lipoprotein envelope which is derived at least in part from the plasma membrane of the host cell, peptide composition of purified chicken embryo membranes was analyzed. In contrast to the virions, the number of peptide units in the host cell membrane were too numerous to give resolution.

A comparison has been made of the sequence of the tyrosine-containing tryptic peptide derived from the purified, unfractionated, lysine-rich histone complement of the calf thymus, rat thymus and calf liver. Two tyrosine-containing peptides, one of which is a fragment of the other, have been obtained from each tissue. The major tyrosine-containing peptide was isolated by a single electrophoretic step in about a 50%, yield. The results indicate that the sequence of amino acids around the single tyrosine present in the lysine-rich molecule is constant from tissue to tissue. However, this sequence varies among the molecular species comprising the lysine-rich histone complement of a single tissue. Within a tissue, a conservative amino acid replacement in the residue penultimate to tyrosine (from the amino terminus) has been detected. The sequence of the tyrosine-containing region was found to be Lys-Ala-Leu-Ala-AlaGly-Gly-Tyr-Asp-Val-Glu-Lys.

Small amounts of peptides were obtained when equal amounts of methane and ammonia were reacted with vaporized aqueous solutions of C14-labeled glycine, L-alanine, L-aspartic acid, L-glutamic acid and L-threonine in the presence of a continuous spark discharge in a 24-hr cyclic process. The experiment was designed to demonstrate the possibility of peptide synthesis under simulated primeval earth conditions. It is theorized that some dehydration-condensation processes may have taken place, with ammonium cyanide, the hydrogencyanide tetramer or aminonitriles as intermediate products, during the early chemical evolution of the earth.

The reaction of NCA's with some amino acids having a nucleophilic functional group on the side chain was studied in a heterogeneous reaction medium (acetonitrile-water). Glutamic acid and aspartic acid, having a free carboxyl group on the side chain, were successfully used to synthesize oligopeptides without interactions of the γ- and β-carboxyl group with NCA's. Two products were obtained by the reaction of NCA with L-lysine, which contains a free amino group on the side chain. e-Protected lysine was used to prepare α-peptides as a nucleophile in the reaction. No racemization was observed in the synthesis of peptides by the NCA method in the heterogeneous solvent system. Oligopeptides with some polar side chains were synthesized by the NCA method.

Improved methods have been devised for the isolation in pmole quantities of a pure flavin pentapeptide and its acid-hydrolysis product (SD-flavin) from inner-membrane preparations of heart mitochondria and from soluble, purified succinate dehydrogenase. SD-flavin differs from riboflavin in still having an amino acid covalently linked to the isoalloxazine ring system. SDflavin may be compared with riboflavin and with various 8n-substituted synthetic flavins by optical spectrophotometry in the neutral and cationic states and by ESR and ENDOR spectrometry in the cationic radical state. On the basis of these experiments is was concluded that the FAD prosthetic group of mitochondria1 succinate dehydrogenase is covalently linked through the 8n-position to the peptide backbone of the protein. This conclusion is in accord with the acid stability of the natural product and its tendency to yield riboflavin under reductive conditions. The unusual pH-fluorescence spectrum of the flavin strongly suggests that the 8n-methylene group is linked to an amino acid through a tertiary nitrogen group.

The amino acid sequences of two rat C-peptides corresponding to two rat proinsulins have been elucidated. Both C-peptides have 31 amino acids. There are differences in two positions, namely, in position 8 where rat-I has a proline and rat-II an alanine residue, and in position 17 where rat-I has a glutamic acid, rat-II a glycine residue. The additional acidic residue in rat-I C-peptide is counteracted by the additional basic residue in the insulin moiety of rat-I proinsulin (lysine B 29), which provides the two proinsulins with the same net charge at neutral pH. The amino acid sequences are: The rat C-peptides contain threonine and arginine, neither of which is present in the C-peptides of man, pig and ox. A comparison of the 5 sequences shows only 10 identical positions out of 31.

Fetal haemoglobin consists of α - and γ-chains. There are two types of human γ-chains:one, (Gγ) in which residue 136 is one of glycine, and another (Aγ), in which that position is oc