scholarly journals The action of proteolytic enzymes on the glycoprotein from pig gastric mucus

1977 ◽  
Vol 163 (2) ◽  
pp. 363-368 ◽  
Author(s):  
M Scawen ◽  
A Allen

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.

1976 ◽  
Vol 159 (3) ◽  
pp. 643-650 ◽  
Author(s):  
A H Gordon

A three-stage method for isolation of α1 macroglobulin and α2 macroglobulin from the serum of normal and injured rats is described. The methods successively used, namely gel filtration, ultracentrifugation and chromatography on DEAE-cellulose, were chosen to minimize loss of tryptic esterase-protecting activity. The two proteins differed slightly with respect to the following properties: mol.wt., α1 macroglobulin 7.46 × 10(5), α2 macroglobulin 7.16 × 10(5); isoelectric focusing, α1, macroglobulin pI 4.4, α2 macroglobulin pI4.5. Amino acid analyses were identical, except with respect to tyrosine: α1 macroglobulin 3.96 ± 0.24, α2 macroglobulin 3.16 ± 0.32 mol/100 mol of total amino acids. When isolated from the serum of uninjured rats, α1 macroglobulin retained the capacity to bind 1.05 mol of trypsin/mol. However, if isolated from serum 2 days after injury only 0.78 mol of trypsin/mol of α1 macroglobulin was bound. α2 macroglobulin isolated from this latter serum bound on average 0.97 mol of trypsin/mol. When reduced with N-acetylcysteine, both molecules formed subunits of size corresponding to that expected for quarter molecules. When α2 macroglobulin was reduced with dithiothreitol, quarter molecules were again produced. α1 macroglobulin, however, when thus treated gave a more complex mixture, containing a component having a mol.wt. of less than 6 × 10(4). Antisera raised against the two proteins permitted estimation of the concentration of each protein in the plasmas or sera of normal and injured rats. Plasma from normal male rats contained 3.76 ± 0.56 mg of α1 macroglobulin/ml (n = 33) and 0.016 ± 0.001 mg of α2 macroglobulin/ml (n=33). After injury by injection of turpentine and cortisone, the concentrations in plasma were at 3 days 5.19 ± 0.81 mg of α1 macroglobulin/ml (n = 12) and at 2 days 1.38 ± 0.35 mg of α2 macroglobulin/ml (n = 12). Antisera to the two proteins did not cross-react with one another. The quarter molecules formed by reduction of both proteins showed increased antigenicity.


1979 ◽  
Vol 42 (05) ◽  
pp. 1652-1660 ◽  
Author(s):  
Francis J Morgan ◽  
Geoffrey S Begg ◽  
Colin N Chesterman

SummaryThe amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser- Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gin- Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys- Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with p-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.


1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.


1976 ◽  
Vol 154 (1) ◽  
pp. 43-48 ◽  
Author(s):  
J D Young ◽  
J C Ellory ◽  
E M Tucker

1. Uptake rates for 23 amino acids were measured for both normal (high-GSH) and GSH-deficient (low-GSH) erythrocytes from Finnish Landrace sheep. 2. Compared with high-GSH cells, low-GSH cells had a markedly diminished permeability to D-alanine, L-alanine, α-amino-n-butyrate, valine, cysteine, serine, threonine, asparagine, lysine and ornithine. Smaller differences were observed for glycine and proline, whereas uptake of the other amino acids was not significantly different in the two cell types.


1963 ◽  
Vol 53 (4) ◽  
pp. 681-713 ◽  
Author(s):  
R. G. Fennah

The feeding of the cacao thrips, Selenothrips rubrocinctus (Giard), on cashew, Anacardium occidentale, one of its host plants in Trinidad, West Indies, is considered in relation to the annual period of maximum population increase on this host and to the choice of feeding sites on individual leaves. On trees observed for three years, populations regularly increased during the dry season, from a low level in December and January to a peak in April or May, and then rapidly declined during the wet season. Even when thrips were most abundant, some trees were free from attack, and this could not be attributed to protective morphological features, to specific repellent substances in the leaf, or to chance. S. rubrocinctus was found to feed on leaves that were subjected to water-stress and to breed only on debilitated trees: the evidence suggested that the adequacy of its supply of nutrients depends on the induction of suitable metabolic conditions within the leaf by water-stress.Both nymphs and adults normally feed on the lower, stomata-bearing surface of the leaf, but in a very humid atmosphere only a weak preference is shown for this surface and if, under natural conditions, it is exposed to insolation by inversion of the leaf, the insects migrate to the other surface. Since the thrips were shown to be indifferent to bodily posture, the observation suggests that their behaviour is governed primarily by avoidance of exposure to undue heat or dryness and only secondarily by the attractiveness of the stomata-bearing surface.Leaves of cashew tend not to become infested while still immature, and become most heavily infested, if at all, soon after they have hardened. Breeding does not occur on senescent leaves. The positions of feeding thrips are almost random on leaves under abnormal water-stress, but otherwise conform to certain patterns that mainly develop in fixed sequence. On reversal of an undetached leaf and consequent transfer of thrips from one surface to the other, there is no appreciable change in their distribution pattern or the apparent acceptability of the substrate. Changes of pattern were readily induced by injury to the plant during a period of water-stress and less easily, or not at all, when water-stress was low. Injury of areas of the leaf by heat was followed by their colonisation by thrips, and partial severance of branches by increased attack on their leaves.Leaves detached from uninfested trees invariably became acceptable for feeding within four hours. During this period, leaf water-content declined and the ratios of soluble-carbohydrate content and α-amino acids to fresh-leaf weight fell slightly and rose considerably, respectively. In the field, the latter ratio was invariably higher for infested than for uninfested leaf tissue, even on portions of the same leaf. If the nutrient value of leaf tissue is determined by the rate at which α-amino acids are extractable through a stylet puncture, the observed change in acceptability for feeding following plucking may be accounted for by the increase in α-amino-acid concentration. Feeding that is restricted on any one tree to the margins of local leaf injuries during prolonged high water-stress and totally absent when stress is low can be correlated with an α-amino-acid content in the living marginal tissue that is high or low, respectively. The ability of thrips to establish themselves and breed on leaves of a particular tree in the dry season and their failure to do so on leaves of the same tree in the wet season conforms with the greater or less amino-acid concentration occurring in the leaf at these respective times.


Author(s):  
George Joseph ◽  
Asha Varughese ◽  
Ann Daniel

Abstract Background A method has been developed and validated for selective, accurate and precise determination of total proteinogenic amino acids and taurine from Infant Formula and Adult/Pediatric Nutritional Formulas (powders, ready-to-feed liquids, and liquid concentrates). The method was reviewed by the AOAC INTERNATIONAL SPIFAN Expert Review Panel (ERP) during the 133rd AOAC Annual Meeting & Expo on September 7, 2019 in Denver, CO, USA and was recommended to First Action Official MethodsSM status. Objective The method involves protein hydrolysis to amino acids, a simple pre-column derivatisation of amino acids and separation of derivatised amino acids by UHPLC. The quantification of amino acids is performed by multi-point calibration using norvaline as the internal standard. The analytical method is capable of quantitative determination for 22 proteinogenic amino acids, but cannot be used to quantitate tryptophan, which is destroyed during the acid hydrolysis step. Asparagine is determined as aspartic acid and glutamine as glutamic acid. The cystine and cysteine are converted to S-2-carboxyethylthiocysteine (CYSx) and the derivative is separated from the other amino acids. Citrulline which is present in some matrices and it is separated from other amino acids is not included in the method performance evaluation in the single laboratory validation (SLV). Method The proposed method met all the performance requirement limits set in standard method performance requirements (SMPR) 2014.013 for total proteinogenic amino acids and taurine. The correlation coefficient of multi-point calibration was not less than 0.999 for any amino acids at any point in the SLV study confirming the validity of linear dynanic range (LDR) and linearity of the method. The individual amino acids in the chromatogram were identified by absolute retention time and relative retention time (RRT) with respect to the internal standard norvaline. There were no significant (S/N Ratio <10) interferences from the reagents or by-products of derivatisation and targeted matrices. The method demonstrated high selectivity. Result Accuracy of the method was validated using standard reference materials (NIST SRM 1869 and 1849a) and spike recovery studies. The amino acid results in the SRMs were within the ranges of Reference Mass Fraction Values. The accuracy of the method was corroboratively validated by spike recovery studies. The average spike recovery range between 93 to 107% ensure the accuracy of the method for amino acids and compliance to the AOAC SMPR 2014.013. Conclusions Precision data of the method demonstrate that it meets the stakeholder requirements as per the SMPR. The mean RSDr for all the amino acids for 17 matrices selected for the SLV were not more than 4%. The method is very sensitive and the LOQ can go down to approximately ten times lower than the SMPR requirements. The sensitivity of method is a direct reflection of its signal to noise ratio which ensures guaranteed method performance at the lower levels of amino acids in these matrices. Highlights Taurine (aminoethane sulfonic acid) unlike the other amino acids is a beta-sulfonic amino acid that is not used in protein synthesis but is found as a free amino acid in tissues. The acidic functional group (-COOH) in common amino acid is replaced with a sulfonic acid (-SO3H) group in Taurine. The method offers baseline separation of citrulline which is an alpha amino acid generally present in Infant Formula and Adult/Pediatric Nutritional products. The separation of citrulline eliminates the risk of interference of this compound with other amino acids. The method can also separate and quantitate hydroxyproline, an important component of collagen that is often used to quantitate collagen. The method is simple and does not include any proprietary chemicals or instruments and can be performed on any basic reverse phase UHPLC system with UV detection.


1962 ◽  
Vol 40 (1) ◽  
pp. 459-469 ◽  
Author(s):  
P. H. Jellinck ◽  
Louise Irwin

Aerobic incubation of estrone-16-C14with peroxidase in the presence of serum albumin and other proteins resulted in the formation of water-soluble, ether-insoluble metabolites in high percentage yields. Similar products were formed when protein was replaced by cysteine or tryptophan but none of the other amino acids tested had any effect. The evidence points to an initial generation of hydrogen peroxide from these nitrogenous compounds by the enzyme acting as an aerobic oxidase, and the subsequent peroxidation of estrone to highly reactive products. These then combine with the protein or amino acid or else undergo alternative reactions. A strong chemical bond is formed with albumin and attempts to release the estrone metabolites from it were unsuccessful. Uterine homogenates from estrogen-treated rats showing high DPNH oxidase activity contained no "peroxidase" as measured by the formation of water-soluble products from estrone in the presence of protein.


1985 ◽  
Vol 40 (7-8) ◽  
pp. 527-530
Author(s):  
Günter Döhler ◽  
Joachim Zink

Abstract The marine diatoms Bellerochea yucatanensis and Thalassiosira rotula were grown at different salinities (20/25, 35, and 40/45‰ salinity (S), respectively) under normal air (0.035 vol.% CO2). No significant variations in the percentage of gross photosynthetic products (e.g. total amino acids, sugar phosphates) were found as a function of salinity during growth. The bulk of the soluble 14C-radioactivity was detected in amino acids. 14C-labelling of glutamine increased markedly with salinity. Low salt - grown algae are characterized by enhanced amino acid pools, mainly of aspartic acid, asparagine and glutamine. It was found that the tested amino acids are not involved in osmoregulation.


1976 ◽  
Vol 230 (4) ◽  
pp. 925-931 ◽  
Author(s):  
RP Forster ◽  
L Goldstein

Little skates, Raja erinacea, and stingrays, Dasyatis americana, were gradually transferred over a period of 4-5 days from full strength to approximatley 50% seawater. Plasma and muscle osmolarity fell. Hematocrits were essentially unchanged. Extracellular fluid volume (ECF) of muscle, estimated as the chloride space, increased 70% during this period. Regulation of muscle cell volume was associated with sharp declines in cellular concentrations of total amino acids (ninhydrin-positive materials) and urea. The osmoregulatory importance of the free amino acid pool in erythrocytes and muscle was a particularly prominent feature in both species. Intracellular amino acid concentration in R. erinacea muscle fell from 214 to 144 mmol/liter during transfer to 50% seawater, urea from 398 to 264, and trimethylamine oxide (TMAO) dropped from 63.9 to 35.8 mmol/liter. TMAO plasma levels were similar in stingray and skate, but muscle TMAO concentrations were much higher in the former. Urea content in stingray plasma greatly exceeded that in R. erinacea-630 and 574 mmol/liter in two specimens-perhaps the highest recorded.


1987 ◽  
Vol 247 (1) ◽  
pp. 15-21 ◽  
Author(s):  
J Kellermann ◽  
C Thelen ◽  
F Lottspeich ◽  
A Henschen ◽  
R Vogel ◽  
...  

The arrangement of the disulphide bridges in human low-Mr kininogen has been elucidated. Low-Mr kininogen contains 18 half-cystine residues forming nine disulphide bridges. The first and the last half-cystine residues of the amino acid sequence form a disulphide loop which spans the heavy- and the light-chain portion of the kininogen molecule. The other 16 half-cystine residues are linked consecutively to form eight loops of 4-20 amino acids; these loops are lined up in the heavy-chain portion of the kininogen molecule. In this way, a particular pattern of disulphide loops is formed which seems to be of critical importance for the inhibitor function of human kininogen.


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