Determination of the arginine content of proteins by the Sakaguchi procedure

The arginine contents of a variety of protein samples determined by the quantitative Sakaguchi method were in good agreement with the values obtained by quantitative ion-exchange chromatography, after acidic hydrolysis. One protein, α-chymotrypsin, while possessing a linear relationship between the colour obtained in the Sakaguchi test and the protein concentration, gave colour yields which corresponded to the apparent presence of an additional Sakaguchi-positive residue. The appearance of this phenomenon was found to be associated with the process of zymogen activation. Subsequently, it was found that two other serine esterases, trypsin and subtilisin, also exhibited this behaviour. Possible reasons for this have been discussed.     

Comparative microphotometric studies of DNA and arginine in plant nuclei

Summary Photometric measurements have been made of the amounts of stain formed in the Feulgen (DNA) and Sakaguchi (arginine) reactions in plant nuclei of differing ploidy.In nuclei of diploid and tetraploid plants of Tradescantia ohioensis and of diploid, triploid and tetraploid plants of Ranunculus ficaria, both Feulgen and Sakaguchi values gave ratios which agreed closely with the ratios of the number of chromatids known to be present. The Feulgen/ Sakaguchi ratio for each of the different types of nuclei measured was very similar both within and between these two species.In the interphase nuclei of five different species, both Feulgen and Sakaguchi values gave bimodal distributions. In the nuclei of differentiating cells, the proportions of values falling into each of the 2C, 4C or 8C classes were the same for both stains.Measurements of the amounts of both stains were made in sequence on the same individual nuclei and a positive correlation found between the two sets of values.In nuclei from differentiating cells of Vicia faba primary roots, the Feulgen/Sakaguchi ratio decreased with increasing distance from the apex.The following suggestions were made from the results: (a) that there is some degree of quantitative constancy of nuclear arginine which parallels that of DNA; (b) that the amount of nuclear arginine, like that of DNA, is doubled during synthesis in interphase; (c) that the syntheses of DNA and arginine in interphase, if not simultaneous, at least occur within the same relatively short period; (d) that there may be a difference in the DNA/arginine ratio between the nuclei of meristematic and differentiating cells.     

Calf thymus histone as a substrate for neutral and acid proteases of leucocyte lysosomes and other proteolytic enzymes

The use of calf thymus histone as a substrate has revealed a previously unknown neutral protease, optimally active at pH 7.2–7.3, in rabbit PMN lysosomes. The Sakaguchi reaction for the colorimetric determination of arginine has been modified, allowing a slow, linear development of color measurable at 500 nm over a period of up to 4 hr at 0–2°C. Specificity for arginine was shown since no other amino acid tested gave any color in the reaction. This new method has been used to measure arginine-reactive hydrolysis products released from histone by PMN lysosomes at neutral pH. Release of tyrosine, measured by the Folin method, was also used as an indicator of hydrolytic activity. Histone proved to be a useful substrate for the acid cathepsins of PMN, comparing favorably with hemoglobin, commonly used to measure such activity. Crystalline trypsin and chymotrypsin also hydrolyzed histone. The kinetics of arginine release by these enzymes over a period of 24 hr resembled those of neutral protease from PMN lysosomes.     

Polypeptide components of the apamin receptor associated with a calcium activated potassium channel

Photoaffinity labeling of rat brain membranes with [125I]ANPAA-apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti-apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to be N-glycosylated. Partial proteolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. These results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but result from cleavage of the 86 kDa polypeptide.     

Quantitative determination of monosubstituted guanidines: A comparative study of different procedures

Analysis of N²-acyl arginine derivatives as well as of arginine analogs lacking in a α-amino function by Weber's modification of the Sakaguchi procedure yielded colored complexes with absorbance values approximately twice that obtained with an equivalent concentration of unmodified arginine. The limitations concerning the applicability of the various modification of the Sakaguchi procedure as well as of fluorimetric assay to the quantitative estimation of a variety of monosubstituted guanidines and proteins are discussed.     

Side effects of reaction media in histochemical blocking procedures

Synopsis 0.2 N NaOH, the reaction medium for 1,2-cyclohexanedione, a specific reagent for arginyl residues in proteins, was found to intensify, at some sites in rat abdominal skin and human gingiva, the Sakaguchi reaction, staining with Pauly's reagent, and anionic dye binding at pH 6.4; at other sites these reactions were reduced, presumably due to extraction of material from sections. 0.2 N NaOH slightly reduced staining after the ninhydrin-Schiff procedure at all sites in rat skin. The interpretation of this finding is obscure, because some sites giving a positive Sakaguchi reaction and staining with anionic dyes failed to stain after the ninhydrin-Schiff procedure. There were also alterations in staining, with the cationic dyes Alcian Blue and Alcian Yellow. It is suggested that 0.2 N NaOH ruptures linkages between polycationic residues of proteins and polanions, demonstrable by Alican Blue. The blockade produced by acetic anhydride-pyridine (4060 v/v) mixtures was stable, in the alkaline conditions required for staining with Pauly's reagent. Pretreatment with pyridine alone reduced tissue binding of anionic dyes.     

Specific modification of fatty acid synthetase from lactating rat mammary gland by chymotrypsin and trypsin.

Fatty acid synthetase from lactating rat mammary gland after limited proteolysis with chymotrypsin or trypsin synthesizes longer chain fatty acids than those produced by the native enzyme. Of the seven partial reactions of the multienzyme complex, only the thioesterase activity was decreased. The results suggest that modification of the fatty acid synthetase product specificity by chymotrypsin and trypsin results from a specific action of these proteases on the thioesterase component. Trypsin, but not chymotrypsin, cleaved a catalytically active thioesterase from the complex; it thus appears that limited trypsinization will be a useful tool for the isolation of the thioesterase component of the multienzyme.     

Sedimentation equilibrium studies of human chymotrypsin II and bovine -chymotrypsin

Sedimentation equilibrium experiments indicate that neither human chymotrypsin II nor bovine δ-chymotrypsin molecules undergo association in the pH range 3–5 where dimerization occurs with α-chymotrypsin. The weight-average molecular weights of human chymotrypsin II and δ-chymotrypsin in a pH 4.4 0.1 ionic strength buffer are 26,200 and 26,400, respectively, using the measured partial specific volumes of 0.722 and 0.727 ml/g at 25 °C. Number-average molecular weight calculations also support the presence of monomeric species at this pH. In the pH range 6–7.6 where sedimentation velocity studies have shown that δ-chymotrypsin associates at concentrations above 3 mg/ml, no association was observed for either the human chymotrypsin II or bovine δ-chymotrypsin in the sedimentation equilibrium experiments where protein concentrations were below 1.2 mg/ml. These studies provide additional evidence that human chymotrypsin II is similar to bovine δ-chymotrypsin.     

Comparative study of the properties of serine proteases of lower and higher vertebrates

Results of the comparative study of trypsin- and chymotrypsin-like serine proteases from pyloric caeca of salmon fishes and trypsin and chymotrypsin of bulls are presented in the paper. The hydrolytic activity of salmon proteases with respect to methyl ethers of N-benzoyl-L-leucine is 2.4 times higher than that of bull chymotrypsin, but with respect to methyl esters of N-benzoyl-L-tyrosine and N-benzoyl-L-arginine the activity of salmon proteases is 6.5 and 80 times lower than that of bull chymotrypsin and trypsin. Salmon proteases in contrast to bull trypsin and chymotrypsin hydrolyze but slightly N-glutaryl-L-phenylalanine para-nitroanilide. It shown that fish proteases are not absolutely specific to synthetic substrates, which is a result of their less pronounced (than in case of bull trypsin and chymotrypsin) differences in structures of binding centres. The study of the salmon protease interaction with some immobilized ligands has confirmed the higher affinity of enzymes to reagents with two space-separated aromatic rings in their composition. It is supposed that salmon proteases interact with such reagents through two sites: hydrophobic "pockets" and probably additional binding site of the active centre. The salmon protease preparation demonstrates higher resistance to inactivating action of formaldehyde within the range of concentrations 2-16% than bull chymotrypsin does.     

Direct physical evidence for stabilization of branched-chain alpha-ketoacid dehydrogenase by thiamin pyrophosphate.

Branched-chain alpha-ketoacid dehydrogenase is a multienzyme complex composed of four subunits. The 46,500-dalton protein is a subunit of the decarboxylase component, which is selectively digested by chymotrypsin. Two peptides of apparent mol. wts. of 36,000 and 15,000 result with loss of enzyme activity. When the complex is saturated with thiamin pyrophosphate and ketoacid substrate, digestion by chymotrypsin does not occur. These data provide direct physical evidence for the stabilization of the complex by the presence of the vitamin B1-derived cofactor.     

FORMATION OF NEW CRYSTALLINE ENZYMES FROM CHYMOTRYPSIN : ISOLATION OF BETA AND GAMMA CHYMOTRYPSIN

A solution of chymotrypsin on slight hydrolysis undergoes an irreversible change into new proteins, two of which are enzymes and have been isolated in crystalline form. The new crystalline enzymes, called beta and gamma chymotrypsins, differ from the original chymotrypsin as well as from each other in many physical and chemical respects, such as molecular weight, crystalline form, solubility, and combining capacity with acid. The new enzymes still possess the same enzymatic properties as chymotrypsin. It thus appears that the irreversible change from chymotrypsin to the new enzymes does not affect the structure responsible for the enzymatic activity of the molecule. The solubility curves of the new enzymes agree approximately with the curves for a solid phase of one component and furnish very good evidence that the preparations represent distinct substances. The various enzymes when mixed at the proper pH have a tendency to form mixed crystals of the solid solution type. Thus at pH 4.0 gamma chymotrypsin combines to form solid solution crystals with either alpha or beta chymotrypsin. Hence at this pH separation of gamma from either alpha or beta by means of fractional crystallization is impossible. At pH 5.0–6.0, however, each material crystallizes in its own characteristic form and at its own rate; thus a fractional separation of the various enzymes from each other becomes feasible.     

Evidence of homology between the β-chain of human haptoglobin and the chymotrypsin family of serine proteases

Amino acid sequences from the β-chain of human haptoglobin are compared with those sequences known for the serine proteases of the chymotrypsin family. In a comparison of some 171 residues of the haptoglobin β-chain (approximately 60% of the protein molecule), approximately 30% of these are identical to residues occurring in sequences of either bovine trypsin, bovine chymotrypsin A, bovine chymotrypsin B, porcine elastase, or bovine thrombin B-chain, and an additional 10% are chemically similar. A combined comparison of the haptoglobin β-chain with the above five serine proteases gave an identity of 56% and a chemical similarity of 11%. Similarity of primary structure is also striking around two of the five half-cystinyl residues so far characterized in long lengths of sequence. These data provide substantial evidence that the β-chain of haptoglobin is homologous to the chymotrypsin family of serine proteases. Proposals are also presented to explain the occurrence of internal homology in the N-terminal region of the β-chain.     

Inhibition of anion transport associated with chymotryptic cleavages of red blood cell band 3 protein

Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS)), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.     

The Role of Hemispheric Functional Specialization in the Analysis of the Relationship between Signal and Nonsignal Information during Image-Shape Classification

The effect of additional information on image shape classification was studied in seven-year-old children. The classified images were either dissimilar in the axis ratio of the basic oval (F) or had an additional element (F + E). In one series of experiments, the additional element was oriented towards a longer axis of the oval, which was a discriminative sign (situation 1, matching); in another series it was rotated by 90° (situation 2, mismatching). The analysis of the successful image classification in series 1 and 2 showed that images with an additional element that mismatches a discriminative sign were classified as being significantly worse than those with matching information. The analysis of the event-related potentials (ERP) from different cortical areas revealed a specific role of the frontal and temporo-parieto-occipital cortical areas in the classification of additional sensory information. The analysis of additional matching information (series 1) was associated with the right hemisphere and evidenced by an increase in N200 wave amplitude in the frontal area and by a negative shift during the development of a late positive complex (250–550 ms) in the temporo-parieto-occipital area. The analysis of additional information mismatching a discriminative sign (series 2) was associated with the left hemisphere and evidenced by an early positive component (P100) in the frontal area, negative components N200–N250, and a further negative shift during the development of late positive complex in the temporo-parieto-occipital area.     

Preparation of photoreactive derivatives of trypsin-chymotrypsin inhibitors from soybeans and chick peas by selective modification of lysine residues

Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas, were prepared by selective modification of the epsilon-amino groups of lysine residues with 2-nitro-4(5)-azidophenylsulfenyl chlorides (2,4(5)-NAPS-C1). The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of the trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.     

Active proteinase inhibitors associated with human breast epithelial cells

The major glycoproteins synthesized by human breast epithelial cells have been characterized [6,8]. The most consistently observed and prominent component in supernatants of organ cultures of breast surgical specimens and of MCF-7 cells was gp 68 which has been immunologically identified as alpha-1-antichymotrypsin (Achy). In the present study we demonstrate that this glycoprotein can form an irreversible complex with chymotrypsin, which indicates that it is a functional inhibitor. The 14C-glucosamine-labeled gp 68 forms a stable, 88,000-dalton, enzyme-inhibitor complex with chymotrypsin. The molecule is secreted continuously for 9 days into a chemically defined, serum-free medium. In addition to the de novo synthesized inhibitor, another component is absorbed from fetal bovine serum and subsequently released into serum-free medium. This component also forms an irreversible, 88,000-dalton complex with enzyme. The observations establish that two types of inhibitors are associated with human breast epithelial cells, one actively synthesized and the other derived from serum. Both of these molecules may have significant roles in stabilizing cell surface components and in protecting extracellular matrices from untimely degradation.     

Digital cows grazing on digital grounds

Picture a pasture open to all (...) As a rational being, each herdsman seeks to maximize his gain. Explicitly or implicitly, more or less consciously, he asks, "What is the utility to me of adding one more animal to the herd?" This utility has one negative and one positive component. 1) The positive component is a function of the increment of one animal. Since the herdsman receives all the proceeds from the sale of the additional animal, the positive utility is nearly +1. 2) The negative component is a function of the additional overgrazing created by one more animal. Since, however, the effects of overgrazing are shared by all the herdsmen, the negative utility for any particular decision-making herdsman is only a fraction of -1. Adding together the component partial utilities, the rational herdsman concludes that the only sensible course for him to pursue is to add another animal to the herd. And another; and another...     

Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria

A higher molecular weight precursor (Mr = 39,000) to the liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = 36,000), is imported and processed by heart mitochondria in vitro in a manner similar to liver mitochondria. In both systems, however, an additional 37-kDa ornithine carbamyltransferase polypeptide appears, but this arises from nonspecific events and, therefore, does not represent a bona fide intermediate in the overall processing sequence. Our experiments demonstrate that the outer mitochondrial membrane of mitochondria contains a protease-sensitive (5 micrograms of trypsin or chymotrypsin/ml, 15 min at 2 degrees C), salt-resistant (1.0 M KCl) protein which is required to maintain import functions. In addition, functional post-translational import requires a component of the reticulocyte lysate (i.e. cytosol) that is used for initially synthesizing precursor enzyme. The component is retained by Sephadex G-25. Import of Sephadex G-25-excluded precursor is restored by fresh reticulocyte lysate but not by a combination of other additives, including Mg2+, K+, ATP, ADP, Pi, succinate, and total translation mixture (minus lysate).     

Purification and characterization of a chymotrypsin inhibitor from the venom of Ophiophagus hannah (King Cobra)

A chymotrypsin inhibitor from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that this protein consists of 58 amino acids, six of these being cysteine residues and is highly homologous to Kunitz-type protease inhibitors. ESI-mass spectrum showed that the protein had a mass of 6493, which is in agreement with that predicted from its primary structure. In contrast to P1 Leu, Met, Phe, Trp, and Tyr appearing in other chymotrypsin inhibitors, a P1 Asn in the novel inhibitor may cause a weak binding (Ki = 3.52 microM) with chymotrypsin. Phylogenetic analysis suggests that the functional variations of the chymotrypsin inhibitor and other Kunitz-type inhibitors probably distinguish from dendrotoxins by accelerated evolution.