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1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

Reduced and carboxymethylated thioredoxin H from Saccharomuces cerevisiae was digested with trypsin and cleaved with cyanogen bromide in Separate experiments. From the amino-acid sequences of isolated peptides after chymotryptic or peptic hydrolysis an alignment of a 17-residue sequence around the redox-active disulfide of thioredoxin II was possible as follows: -Phe-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys-Met-Ile-Ala-Pro-Met-Ile-Glu-Lys- Comparison of this structure with the corresponding amino-acid sequence of the thioredoxin from Escherichia coli B showed identical residues in 10 consecutive positions which included the tryptophan residue, the disulfide ring and the five residues following this on the COOH- terminal side. The COOH-terminal sequence of yeast thioredoxin II was: -Glu-Ala-Ile-Ala-Ser-Asn-Val and the NH2-terminal residue was valine. The sequence results indicated a considerable homology in primary structure between yeast and E. coli thioredoxins and suggested the existence of a common ancestral gene for thioredoxins. The sequence of a peptide from the active site of yeast thioredoxin I showed that the structure around the disulfide bridge was: -Tyr-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys-. This indicated that thioredoxin I and II from yeast have different primary structures. [ABSTRACT FROM AUTHOR]

The study of the structure of the peptides arising from native, oxidized or reduced and maleinized bovine growth hormone on incubation with trypsin, ehymotrypsin and pepsin is reported. The data obtained permitted the assembly of a unique sequence of amino acids for the poly-peptide chain of the protein. Various corrections and one addition to the sequence previously communicated are made. [ABSTRACT FROM AUTHOR]

Bence-Jones protein DEL was isolated from the urine of a patient with multiple myeloma by ammonium sulfate precipitation. Ion-exchange chromatography provided no further purification of the protein; the ammonium sulfate precipitate consisted of only one protein moiety as tested by polyacrylamide electrophoresis. Protein DEL contains 213 amino acids and has a molecular weight of 22500. Eighteen peptides have been isolated by ion-exchange chromatography after tryptic digestion of the completely reduced and aminoethylated protein. Sequence studies were performed mainly on these tryptic peptides. In addition, 14 chymotryptic pepfides were isolated and characterized to obtain overlapping peptides for the whole V-region and for some tryptic peptides of the C-region. Protein DEL is related to subgroups III and IV of the γ-chains as indicated by a deletion of three amino acids after position 27, a common pattern of these two subgroups. Furthermore, position one is deleted in protein DEL as in the other proteins of subgroups IH and IV but in contrast to subgroups I and II of the γ-chains. However, marked differences exist between protein DEL and the proteins of subgroups IH and IV. a) The deletion of two amino acids in positions 95 and 96, characteristic of subgroup IV, was not found in protein DEL. b) 19 of the 27 linked exchanges specific for subgroup IV are substituted in protein DEL. Although the insertion of two amino acids after position 94 is the same as in subgroup III, a high exchange rate was observed for proteins DEL and Sh. Therefore it is concluded that protein DEL does not belong to either subgroups III or IV, but is the first example of a new subgroup of the γ-chains. [ABSTRACT FROM AUTHOR]

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 filtration 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 rune 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. [ABSTRACT FROM AUTHOR]

To clarify the functional role of amino acid residues in copper binding and in the maintenance of ceruloplasmin catalytic activity, the protein was chemically modified by iodination. It is shown that addition of 20 mol iodine per 1 tool ceruloplasmin mainly produces iodination of tyrosine residues, while the protein modification by higher iodine concentrations (60 tool and more per 1 mol protein) leads also to iodination of histidine. Iodination of ceruloplasmin is followed by loss of copper and the oxidase activity. There is a correlation between copper loss and ceruloplasmin catalytic activity and iodinated histidine content at high concentrations of iodine in the medium. It appears that tyrosine residues are involved in maintaining the active conformational state of ceruloplasmin, while histidine residues directly bind copper in its catalytic center. Peptide maps for 131I-labeled ceruloplasmin show that most tyrosine peptides of the protein iodinated at small iodine concentrations are localized in the region of acid and alkaline peptides. Histidine peptides, whose iodination in accompanied by decrease in the oxidase activity and copper content, are mainly localized in the neutral peptide region. One of the neutral peptides having a well-defined Pauly reaction was isolated in a pure form. N- and C-terminal amino acids of the peptide and its amino acid composition were determined. [ABSTRACT FROM AUTHOR]

The ethanol-active isoenzyme of alcohol dchydrogenase from horse liver has been carboxymethylated with iodo[2-14C]acetate in 6 M guanidine hydrochloride. A study of the tryptic digest of the carboxymethylated enzyme has shown that it contains 14 unique sequences with [2-14C]carboxymethylcysteine, two with tryptophan, one with C-terminal phenylalanine and one with a blocked N-terminal residue which represents the X-terminal sequence in the protein chain. This number of unique residues found m peptides is in each ease half the number previously found by direct amino acid analysis (based on a molecular weight of 80 000) of the intact enzyme. These results support the view that the native isoenzyme is a dimer consisting of two similar and probably identical polypeptide chains each containing about 370 amino acid residues. Failure to demonstrate the presence of more than one type of chain by chromatography of the carboxymethylated enzyme on DEAE-cellulose provides additional support for the dimer hypothesis. An alternative proposal [13] that the enzyme is composed of two pairs of dissimilar chains each with a molecular weight of 20 000 appears to be incompatible with these results. [ABSTRACT FROM AUTHOR]

The reaction rates between dansyl chloride and water, amino acids or peptides are studied as a function of pH and temperature. The rate of hydrolysis of dansyl chloride is constant and low up to pH 9.5 and above this pH it increases rapidly. The various reactive groups of amino acids and peptides react with dansyl chloride in their unprotonated form. It is shown that a compromise for optima conditions of dansylation (pH and temperature) may be found, taking into account the rate of hydrolysis and the pk of the group to be dansylated. A complete chromatographie separation on Silica gel G thin layer plates of dansyl amino acids is proposed using 4 solvents. The quantitative recovery of the separated derivatives is described. The study of the acid hydrolysis of dansylated proteins shows that the release of the dansyl derivatives from the peptide chain is faster than that of free amino acids; it also shows that the destruction of dansyl amino acids occurs rather rapidiy. Therefore a hydrolysis time of 4 hours (110°) is suggested, except in the special cases of the release of dansyl valine, dansyl leucine or dansyl isoleucine which needs 18 hours of hydrolysis. For quantitative estimation of each dansyl derivative a correction factor is given, taking into account the loss during hydrolysis, the recovery from the plates and the individual fluorescence of any dansyl derivative as compared to the fluorescence of a reference compound. The general conditions described in this work require 1 nmole of material for qualitative determination of the N-terminal amino acids, and 5-10 nmoles for their quantitative estimation. Some examples (α-chymotrypsin, trypsinogen, ribonuclease, lysozyme aspartokinase and triose phosphate dehydrogenase) illustrate the efficiency of the method. [ABSTRACT FROM AUTHOR]

A new procedure for the determination of the N-terminal groups in peptides and proteins is described. The free amino-groups are allowed to react with 2-fluoro-3-nitropyridine or 2-fluoro- 5-nitropyridine in slightly alkaline solution, the resulting derivatives are easily hydrolyzed in dilute hydrochloric acid in sealed tubes to give the corresponding nitropyridyl amino acids. Most of these compounds are quantitatively recovered under these conditions; in particular it is possible to estimate proline and glycine as N-terminal residues. After isolation the nitropyridyl amino acids can be estimated spectrophotometrically; in some cases it is possible to regenerate the parent amino acid by treating the derivatives with strongly alkaline solutions. The adequacy of the present techniques in relation to those of other workers was tested on insulin, lysozyme and aldolase; the advantages and peculiarities in comparison with these procedures are discussed. [ABSTRACT FROM AUTHOR]

The complex protein carrying the two threonine sensitive activities aspartokinase I and homoserine dehydrogenase I is composed of six subunits of a molecular weight 60,000. Furthermore, quantitative determination of the N-terminal amino acids indicates the presence of six methionine residues per mole of native enzyme (mol wt, 360,000). Equilibrium sedimentation studies of N-ethylmaleimide treated protein shows that its six disulfide bridges, revealed by chemical analysis, are intra-chain. Fingerprints of tryptic digests of the protein fail to reveal any difference between the subunits. [ABSTRACT FROM AUTHOR]

The complex protein carrying the two threonine sensitive activities aspartokinase I and homoserine dehydrogenase I has been obtained in a homogeneous state from extracts of Eseherichia coli K 12. The molecular weight of the protein has been determined by sedimentation equilibrium to be 360,000 dalton. The amino acid composition is given. Titration of sulfhydryl groups by 5-5'-dithiobis- (2-nitrobenzoic)acid leads to an estimation of 16 to 18 reactive cysteines per mole of the protein, all of which can be protected by addition of the allosteric inhibitor, L-threonine. [ABSTRACT FROM AUTHOR]

Isolated cyanogen bromide fragments of the αB2 chain of α-crystallin contained 67 and 107 residues. respectively. Part of the sequence of the C-terminal fragment was elucidated by Edman and Begg sequenator analysis. Sequences of the tryptic peptides were determined by a combination of various Edman degradation techniques. Overlaps of tryptic peptides were established by selective modification of lysine residues and subsequent tryptic cleavage and by isolation of peptides after hydrolysis with chymotrypsin, thermolysin and a staphylococcal protease. The sequence of the αB2 chain comprises 175 residues corresponding to a molecular weight of 20070. This value is somewhat lower than that assumed hitherto. A high degree of homology between the αB2 and the αA2 chain, the major polypeptides, of bovine α-crystallin was observed. [ABSTRACT FROM AUTHOR]

Previous results on RNA-labelled nuclear particles were compatible with a folded ribonucleo- protein strand structure. In the present experiments, particles were labelled with tritiated amino acids or with tritiated amino acids and [32P]orthophosphate in vivo; they were prepared by sonication of nuclei. 1. Proteins were released progressively from the particles (ϱ = 1.39 g/ml) as shown by the accumulation of material of ϱ = 1.31 -1.35 g/ml after treatment with 0.25 M and 0.4 M NaCl. Concomitantly, high-density ribonucleoproteins were formed. 2. Proteins released from the particles at various NaCI concentrations (up to 2 M) sedimented between 0 and 15 S. 30-S aggregates corresponding to "informofers" were never detected. Some proteins remained bound to the RNA even after treatment with 2 M NaCI. 3. Large protein aggregates contaminated the smallest particles (up to 50-60 S) as shown by the sedimentation properties of the products of extensive ribonuclease digestion and the density of protein-labelled fractions. 4. About half of the protein released from the particles by pancreatic ribonuclease treatment had a low sedimentation coefficient (0-15 S) after formaldehyde fixation. The other half had higher polydisperse sedimentation coefficient, and possibly resulted from artificial cross-linking. A 30-S protein peak was not detected. 5. The material released from the particles by ribonuclease banded at ϱ = 1.35 g/ml, a higher density than that of free proteins (ϱ = 1.31 g/ml). Treatment with 1 M NaCI after ribonuclease digestion released free proteins, some of which were phosphoproteins (ϱ = 1.31 g/ml) and ribonucleo- proteins of higher density. It is suggested that these complexes contain ribonuclease-resistant RNA. These results confirm and complete our previous ones. They are not compatible with the informofer model of the monoparticle since 30-S protein aggregates could not be isolated after salt or ribonuclease treatment. They are consistent with the folded ribonucleoprotein strand model which predicts the sequential release of proteins. [ABSTRACT FROM AUTHOR]

Histone F3 has been fragmented into 10 non-overlapping fragments. With the aid of the automatic Edman degradation procedure for proteins using 3-(N,N-dimethylamino)propyne buffer the first 48 amino-acid residues in the uncleared protein have been positioned. The remainder of the amino-acid sequence of this protein was elucidated by subjecting the aligned peptides to the automatic degradation. The amino acids obtained during the degradation were quantitated and identified by gas chromatography and amino-acid analysis. The primary structure of this histone is compared to the corresponding fraction from calf which had been independently determined by another laboratory. [ABSTRACT FROM AUTHOR]

Histidine decarboxylase from Micrococcus sp.n. was carboxymethylated and two types of subunits, I and II, were separated in excellent yield by exclusion chromatography on Sephadex G-100 in 8 M urea. Total compositions, number of tryptic peptides and N-terminal groups of the subunits were analyzed. The larger subunit I is composed of all amino acids commonly found in proteins and has a blocked N-terminal residue, whereas the smaller subunit II does not contain proline, histidine or cysteine and has N-terminal methionine. The number of tryptic peptides detected in peptide mapping experiments of subunit I is about 27 and that of subunit II is about 15. These figures are in good agreement with the total number of lysine and arginine residues determined by amino acid analysis, if subunit I contains about 270 residues, corresponding to a molecular weight of 29000, and subunit II about 70 residues, corresponding to a molecular weight of 7000. It is concluded that histidine decarboxylase contains two apparently homogeneous but completely different polypeptide chains and that the native enzyme of molecular weight 110000 is a hexamer with three subunits of each type. [ABSTRACT FROM AUTHOR]

The primary structure of a non-immunoglobulin amyloid protein AS has been determined. The protein was found to consist of 76 amino acid residues corresponding to a molecular weight of 9145. The sequence analysis showed clearly that the protein was homogeneous. A characteristic distribution of hydrophobic amino acids was observed and suggested as being of importance for the ability of this protein to form fibrils. A comparison of the protein with other amyloid protein AS showed a high degree of variability, particularly in the carboxyl-terminal region. [ABSTRACT FROM AUTHOR]

The crystal protein of Bacillus thuringiensis var. tolworthi has been separated into two polypeptide chains having molecular weights of 55000 (A) and 120000 (B). The ratio of A to B in the crystal appears to be 2:1 on a molar basis. Differences in amino-acid composition and biological activity exclude the possibility that the larger polypeptide chain is a dimer of the smaller one. The full toxicity of the unfractionated protein to larvae of Pieris brassicae is retained by fraction A, whereas fraction B is non-toxic. [ABSTRACT FROM AUTHOR]

The determination of the amino acid sequence of the C-peptide of human proinsulin is presented. Two constituent fragments of C-peptide, Chy 1+ 2 and Chy 3 (residues 1-24 amd 25-31, respectively) were liberated by mild chymotryptic digestion and isolated gel filtration and ion exchange chromatography. From Chy 1 + 2 was obtained seven smaller peptides by further digestion with chymotrypsin. The sequence analysis of these fragments allowed an overall structure to be assigned. Papain digestion of the intact C-peptide provided 12 peptides, the compositions of which provided unequivocal confirmation for the proposed sequence. [ABSTRACT FROM AUTHOR]

The atomic structure of the monomeric insect haemoglobin is closely similar to the structure of whale myoglobin, although only about 20% of the amino acids are identical in both protein molecules. A quantitative comparison of both structures, however, showed that in the vicinity of some amino acid substitutions considerable changes occur. The comparison also revealed unexpected long range structural changes induced by amino acid deletions. In particular a deletion of four amino acids in the A B corner shifts the whole helix B considerably. The general distribution of amino acids in erythrocruorin is analogous to that which has been found m other haemoglobin molecules. The large proportion of phenylalanines in the hydrophobic core of the molecule is remarkable. Also the haem pocket is lined with seven phenylalanine side chains. Four of these are unknown in vertebrate haemoglobins. The distal histidine E7, which is invariant in all known vertebrate haemoglobins, is replaced by a glutamic acid, which has no haem contact, but protrudes into the surrounding solution. This proves that a histidine or another polar residue is not essential for the function of a haemoglobin molecule as a reversible oxygen carrier. [ABSTRACT FROM AUTHOR]

The structures of nine avian ovalbumins were compared by search gel electrophoresis, ultracentrifugation, tryptic peptide mobillties, and amino acid composition. The results showed that the ovalbumins belonged to two structurally different groups which corresponded to the two taxonomic orders studied. Relationships within the orders were also indicated. [ABSTRACT FROM AUTHOR]

Initial velocity kinetics of the reverse direction showed that the saccharopine dehydrogenasecatalyzed reaction was consistent with a mechanism that the addition and release of reactants to and from the enzyme follow an obligatory order. The product inhibition studies in the forward direction also revealed kinetic patterns consistent with the ordered mechanism where the sequence of addition of substrates being NAD+ and saccharopine, and that of release of products being lysine, α-ketoglutarate and NADH. The various kinetic parameters determined at pH 6.8 and 22° are aisc described. [ABSTRACT FROM AUTHOR]

Molecular fragments of various lengths arc formed by treating acid-soluble collagen with collagenase from Clostridium histolyticum. Fragments 78, 105 and 125 nm long can be isolated by fractional ammonium sulphate precipitation. Their position in the collagen molecule has been determined by electron optical investigations. Denaturation of the individual fragments yielded peptides of the α1- and α2-chains which could be separated by CM-cellulose chromatography. These peptides, α1 (78) and α1 (105), are in the region of two of those obtained by cyanogen bromide cleavage, namely, α1-CB7 and CB6; α1 (125) is superimposed on α1-CB3, CB7 and CB6. In order to obtain smaller fragments of these CB peptides of the α1-chain, the three, fragments from collagenase treatment were cleaved further with cyanogen bromide and the resultant pepfides were isolated and characterized. Their position along the α1-chain was determined electron microscopically after renaturation to the triple helical structure. Each fragment of the α1-chain yields two chromatographically and electrophoretically distinguishable peptides which have been designated α1 and α1*. The difference between the two was found to reside in the—COOH end of the molecule: the α1* peptide contains seven amino acids more than the α1. [ABSTRACT FROM AUTHOR]

Penicilloylated proteins which are found as an impurity in 6-aminopenicillanic acid can be exhaustively digested by pronase to yield amino acids and small peptides. This degradation converts the point polyvalent antigens into a mixture of mostly monovalent haptens which are much less immunogenic and less capable of eliciting immune reactions in sensitized animals. The degradation of the protein impurity can be carded out in 6-aminopenicillanic acid preparations, since even a large excess of 6-aminopenicillanic acid does not appreciably inactivate or inhibit the enzyme. In order to avoid the contamination of 6-aminopenicillanic acid with a proteolytic enzyme, pronase was converted into a water insoluble form by coupling it with bromoacetyl cellulose or carboxymethyl Sephadex. These insoluble derivatives of pronase were found to retain their activity and broad specificity. Thus, they can be readily removed from the medium upon completion of the impurity degradation, to be used repeatedly in a continuous process. [ABSTRACT FROM AUTHOR]

The metabolism and physical-chemical properties of nascent(precursor) 40 and 60 S sub-ribosomal particles have been studied in exponentially growing Tetrahymena pryrifomis GL. Analysis of formaldehyde-fixed 40 and 60S particles in isopyenic CsCl gradients reveals that they have a lower buoyant density than the corresponding 30 and 50 S ribosomal sub-units. More over, overall labeling of the cells with [3H]amino acid and [32P]phosphate shows that the precursor particles attain larger 3H/32P ratios than the corresponding sub-units, indicating that the precursor particles contain extra(accessory) protein that splits off when they are converted into ribosomal sub-units. The extra protein constitute 10 to 20 % of the total structure protein. Experiments in pulse-labeling with radioactive amino acids indicate that 40 and 60 S particles are very rapidly labeled. This suggests that, apart from the RNA moiety, at least parts of the structure ribosomal protein moiety associated with the nascent particles is newly synthesized. The possible involvement of 40 and 60 S particles in sub-unit exchange between rounds of translation was investigated by comparing their kinetics of labeling with that of the ribosomal sub-units. The results of the comparison indicate that the rate of recycling of ribosomal sub-units through the pool of 40 and 60 S particles is too slow account for sub-unit exchange between each round of translation. [ABSTRACT FROM AUTHOR]

Absorption spectroscopy and circular dichroism measurements have been used to elucidate thc mechanism of lysozyme inactivation on photo-oxidation of the methionyl residues to methionine sulphoxide. The monosulphoxide derivative, which is formed by irradiation of the protein in aqueous solution, displayed spectral properties very similar to those of native lysozyme and retained about 54% enzyme activity. This reduced catalytic efficiency appeared to be due to limited conformational changes of the protein molecule. Striking differences were found, on the contrary, between the optical parameters of native lysozyme and of the di-sulphoxide derivative, which is prepared by irradiation in acetic acid solution and is almost devoid of enzyme activity. Studies on the unirradiated lysozyme demonstrated that this extensive denaturation was caused by the action of the protic solvent used. Whereas the acid-induced denaturation was reversible in the case of unmodified lysozyme, the photo-oxidation of the two methionyl residues irreversibly blocked the molecule in a largely disordered form. These results are discussed with respect to the location of the two methionines in the known atomic model of lysozyme. [ABSTRACT FROM AUTHOR]

It is shown that the perturbation of the ultraviolet absorption spectra of the tyrosine and tryptophan chromophores in aqueous solutions by addition of solvents of lower polarity leads not only to red shifts but also to changes in peak intensity, and oscillator strength. The effect is much greater in tyrosine than in tryptophan, and can be as large as 30% (transfer from water to ethanol). It is shown that observed difference spectra can be reasonably reproduced by the addition of two calculated curves, one corresponding to a shift, the other to a change in intensity, i. e. an admixture of shift difference spectrum and absolute spectrum. Similar intensity changes are seen to accompany the denaturation of several proteins. The changes of the characteristic parameters of difference spectra with the concentration of perturbant are analyzed. The relation between the difference spectra generated by aromatic chromophores near 230 mμ. and those m the 280 mμ. region are considered, and their relative magnitudes have been estimated for a variety of perturbants. It is shown that in denaturation. of a number of proteins, the difference spectra near 230 mμ cannot be satisfactorily accounted for solely in terms of the model compounds containing aromatic chromophores. Possible contributions to difference spectra of proteins in this region are considered. [ABSTRACT FROM AUTHOR]

The hemoglobins of a child homozygous for the Leporeboston gene have been analyzed. Only hemoglobin F and Lepore were observed. Hemoglobin Lepore was isolated and the peptide chains of this hemoglobin were separated. The amino acid composition of the Lepore chain and of the tryptic peptides obtained from this chain was determined. The peptides isolated accounted for the amino acid composition of the Lepore chain and indicated that this chain is 146 amino acid long. The switch region from the δ-like sequence of the Lepore chain to the β-like sequence has been localized between residue 87 and residue 116. [ABSTRACT FROM AUTHOR]

Three forms of α-lactalbumin have been isolated from pooled cows' milk: α-lactalbumin B, glyco-α-lactalbumin B and a new form of α-lactalbumin, α-lactalbumin III. α-Lactalbumin III accounts for about 5% of the total α=lactalbumin of the milk. This protein has very similar physical and kinetic properties to α-lactalbumin B but its amino acid composition is different; in particular it has only three disulphide bonds rather than the four usually found in α-lactalbumin. [ABSTRACT FROM AUTHOR]

The impact of protein nutrition upon serine biosynthesis and catabolism in rat liver was investigated by determining the activity of two key enzymes in the pathway of serine metabolism, phosphoglycerate dehydrogenase on the pathway leading from pyruvate to serine, and serine dehydratase on the catabolic way transforming serine to pyruvate. It was found that increasing the protein content of the diet induces serine dehydratase and represses reciprocally phosphoglycerate dehydrogenase. For casein, a protein of high nutritive value, the activities of both enzymes are at equilibrium at the 20% dietary protein level, which is near the optimum casein level for rat growth. With gluten, a protein of low nutritive value, equilibrium is reached at almost the same dietary protein level (22%), although optimal growth is not attained prior to the 50% dietary protein level. In this comparison, there is no correlation between the nutritive value of the proteins (growth measurement) and their regulatory action on the two enzymes tested. Extension of the studies to other proteins, such as egg, showed however that egg, the protein of highest nutritive value, has also the greatest effect on enzyme regulation. The conclusion is that a certain relationship between nutritive value and enzyme-regulatory action exists, but that the determining factors (amino acids) are partly different. The role of individual dietary amino acids upon serine metabolism was therefore investigated. It was first shown that only the essential amino acids as a group were responsible for the reciprocal induction and repression of the two enzymes. By feeding amino acid mixtures lacking one essential amino acid at a time to the animals, we established subsequently that four essential amino acids only, i.e. methionine, tryptophan, threonine and valine are active in regulating the two enzymes. Cystine influences only phosphoglycerate dehydrogenase. The enzyme-regulating action of proteins can thus be expressed in terms of their content in “active” amino acids, whereas their nutritive value depends on the presence of all essential amino acids. The similar behaviour of casein and gluten in enzyme regulation in spite of the great difference in nutritive value is thus fully explained, since the deficient amino acid in gluten, i.e. lysine, is not active in the regulation of the two enzymes studied. Finally a coherent theory for the reciprocal serine dehydratase repression and phosphoglycerate dehydrogenase induction in rat liver by lack of an “active” amino acid is proposed. It is based on the presence of a posttranscriptional repressor which both inhibits messenger translation and promotes messenger degradation, and on the fact that the translation of small proteins is less impeded by amino acid deficiency than that of bigger polypeptide chains. [ABSTRACT FROM AUTHOR]

In a simplified procedure for isolation of phoratoxin, the dried plant material (stems and leaves) was extracted with 2% aqueous acetic acid. The exact was treated with polyamide to remove colored impurities and subjected to gel filtration on Sephadex G-25. The high-molecular-weight fraction was chromatographed on SE-Sephadex, using a gradient elution technique, whereby pure phoratoxin was obtained. The mobile was homogeneous by standard criteria of poem purity. The molecular weight, determined in the ultracentrifuge by the sedimentation equilibrium method, is 5000. Phoratoxin has the following amino acid composition (residues/mole): alanine 2, arginine 3, aspartic acid 4, &frac12-cystine 6, glycine 6, histidine 1, Isoleucine 3, leucine 1, lysine 4, phenylalanine 1, proline 2, serine 5, threonine 5, tryptophan 1, tyrosine 1, valine 1. The molecular weight, calculated from the amino acid composition, is 4884. The material contains no reducing sugars, amino sugars or sialic acids. The LD50, determined by intraperitoneal injection m mice, is 0.57 ± 0.05 mg/kg bodyweight. [ABSTRACT FROM AUTHOR]

Pepsin has been modified by reaction with [14C]trimethyloxonium fluoroborate at pH 5.0. The loss of catalytic activity consequent upon modification can be ascribed to the esterification of carboxyl groups in the protein. The relationship between the number of methyl groups incorporated and the remaining catalytic activity provides clear evidence that at least two carboxyl groups are essential to the activity of pepsin. Mechanistic formulations that require two carboxyl groups now rest on firmer ground. [ABSTRACT FROM AUTHOR]

The purification and chemical characterization of three Bence-Jones proteins and one light chain of an IgG myeloma protein of the rat strain LOU/Wsl are described. The amino acid composition of the four chains is very similar but not identical. Fingerprint analyses show a pattern of peptides common to all four proteins as well as unique features. The compositions of the common peptides are given. The data indicate that the four light chains are of the kappa type. [ABSTRACT FROM AUTHOR]

Mitochondria purified from streptomycin-bleached mutant SB3 (Krawiec and Eisenstadt, 1970) of Euglena grades Klebs strain z, were found to be active in incorporation in vitro of labeled amino acids into proteins. Chloramphenicol and puromycin were most effective inhibitors for the system, while cycloheximide has very tittle effect. Incorporation of the amino adds was resistant to added DNAase and RNAase. tRNA and tRNA synthetases obtained from mitochondrial fractions were active in vitro and were found to be different from those obtained from chloroplasts of green Euglena gracilis for the amino acids tested. [ABSTRACT FROM AUTHOR]

By peptic or tryptic proteolysis of different fragments of bovine encephalitogenic protein, a set of small fragments covering the following regions of the native protein have been obtained: 44-68, 44-89, 75-91, 93-116, 114-130, 134-150, and 154-170 (the amino acid residues are numbered according to [1]). These fragments were used in the macrophage migration inhibition technique in guinea pigs (an assay which is vividly considered as a correlate in vitro to delayed type hypersensitivity) to study the localization of antigenic determinants in the protein. The results indicate the presence of at least one determinant in each of regions 44-68, 134-140. and 104-170. Furthermore, the rather strong determinant, which has previously been localized around the single tryptophan residue in the protein does not seem to be contained within peptide 114-130, as this peptide is considered unreactive in the assay. On the other hand, this peptide shows full encephalitogenic activity. [ABSTRACT FROM AUTHOR]