Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Purified bovine beta-casein was digested in vitro with varying mixtures of purified proteinases and peptidases including trypsin, chymotrypsin, dipeptidyl peptidase IV (DP IV), aminopeptidase M and prolidase. In digestion mixtures without DP IV the yield of free amino acids was considerably lower than in the corresponding assays with this peptidase. Especially, the release of proline increases drastically from almost zero to the theoretical amount in the presence of DP IV. Quantitative results indicated that the specificities of the two microvillar peptidases (aminopeptidase M and DP IV) optimally complemented each other. This effect elucidates the hitherto obscure physiological role of intestinal DP IV. A similar effect may also apply to other caseins and nutritional proteins.
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PMID:Complementary action of dipeptidyl peptidase IV and aminopeptidase M in the digestion of beta-casein. 287 57

A new peptide of 20,000 daltons was found in human milk as a constituent of the casein micelle. Enzymic digestion with plasmin or trypsin revealed that the peptide was identical with a degradation product of human beta-casein. The amino acid composition of the degradation product and the previously reported sequence in the N-terminal region of human beta-casein suggested that the peptide was a fragment of beta-casein lacking the C-terminal region. The thermal sensitivity of this peptide was higher than that of beta-casein, but the peptide lost the property of calcium-dependent precipitation, which intact beta-casein possesses.
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PMID:A 20,000-dalton casein fragment in human milk. 293 32

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.
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PMID:Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs. 390 77

Proteolytic activity in mastitic skim-milk was often 5-10 fold higher than in normal milk, its level being related to somatic cell count but not precisely correlated with it. In milks with the highest levels of activity plasmin accounted for about one third of the total proteinase. A further third was sedimented with the micellar fraction together with the plasmin, but unlike plasmin, was not inhibited by addition of soyabean trypsin inhibitor (SBTI). The final third remained in the serum phase. Polyacrylamide gel electrophoresis (PAGE) showed that alpha-sl- and beta-caseins were degraded at about the same overall rate. The plasmin produced the usual readily identified fragments from beta-casein, but incubation of mastitic milk also produced changes in patterns in the gamma-casein region differing from plasmin-induced changes, which were also apparent when the micellar fraction was incubated. As they were inhibited by SBTI, a second trypsin-like enzyme in addition to plasmin may also have been present. Other proteinase(s) not inhibited by SBTI was also associated with casein micelles and produced at least 3 characteristic protein fragments seen on PAGE. The serum phase proteinase(s) was likewise not inhibited by SBTI, and did not produce any well-defined electrophoretic bands, suggesting a rather non-specific breakdown of caseins. After separation of mastitic whole milk, a considerable proportion of the proteolytic activity was found in the cream phase. The proportion was enhanced by freezing and thawing, and the enzyme appeared to be identical to the SBTI-resistant micellar proteinase. Because of the considerable proteolysis likely to occur under the time and temperature conditions involved, our results may provide some explanation for the problems encountered in cheesemaking with mastitic milks (e.g. yield losses, poor curd strength and off-flavour development).
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PMID:Qualitative and quantitative determination of proteolysis in mastitic milks. 621 34

1. The protein kinase activity associated with the phosphoprotein fraction of calf thymus nuclei has been examined for its ability to phosphorylate exogenous phosphoprotein substrates. beta-Casein and phosvitin are both efficient phosphate acceptors. Phosphorylation of alpha s1 and beta-caseins was shown, directly, to occur at threonyl-49 and threonyl-41 respectively. Both threonyl residues occur within the sequence Thr-Glu-Asp. 2. alpha s1-Casein becomes a much better substrate for the kinase when partially dephosphorylated. A similar response is shown by a phosphopeptide containing the alpha s1-casein phosphate cluster and is due to a new phosphorylation site becoming available. Efficient phosphorylation of beta-casein requires that the phosphate cluster (residues 15-19) be intact and results are presented which are consistent with there being a similar requirement for phosphorylation of the site created in alpha s1-casein by partial dephosphorylation. 3. Comparison of genetic variants of beta-casein as phosphate acceptors for the kinase shows that the presence of lysyl residues close to the phosphorylation site markedly depresses the rate of phosphorylation. Maleylation of beta-casein increases the rate of phosphorylation for all of the variants tested, although to varying extents. Treatment of maleylated beta-casein with trypsin to remove the N-terminal phosphopeptide inhibits phosphorylation by the kinase. 4. The structural determinants of beta-casein allowing it to be efficiently phosphorylated by the kinase are concluded to be the presence of a sequence surrounding the phosphorylation site, which is rich in acidic amino acid residues and from which basic residues are absent. The acidic phosphate cluster of beta-casein also promotes phosphorylation either by interacting directly with the enzyme or through its influence on the conformation of beta-casein.
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PMID:Substrate specificity studies on a calf thymus nuclear phosphoprotein kinase. 628 3

One of the major actions of prolactin in the mammary cell is to activate the expression of casein genes by enhancing the transcription rate of the genes. Anti-prolactin receptor antibodies can mimic prolactin action when added to mammary cells, suggesting that a relay is formed at the membrane level and transferred to the target genes. None of the classical hormone intracellular relays can account for the transfer of the prolactin message. The incubation of membranes from various tissues containing prolactin receptor with prolactin provokes the release of a factor which specifically accelerates the transcription of the beta-casein gene in isolated mammary nuclei. The factor is thermostable, inactivated by trypsin and is eluted from G-10 Sephadex as a molecule smaller than 1000 daltons. The action of the factor is prevented by phosphatase inhibitors. These results are strikingly reminiscent of those obtained with insulin which activates enzymes via a factor released from the membranes and which dephosphorylates the enzymes. The analogy between the action mechanism of prolactin and insulin is discussed.
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PMID:[Comparison of the mechanism of action of insulin and prolactin]. 630 40

(1) High-resolution 31P-NMR was used to study the environment of the phosphoserine residues of the phosphoproteins, alpha s1-casein B, beta-casein A2 and beta-casein C. For reference purposes 31P-NMR spectra of phosvitin and ovalbumin were also collected. (2) 31P resonances were assigned to specific phosphoserine residues as a result of comparisons of the high-resolution 31P-NMR spectra for alpha s1- and beta-caseins and for peptide fragments of these proteins obtained by cyanogen bromide and trypsin cleavage. (3) Measurements of the enhancement of the relaxation rate for water protons (1H) on addition of Mn2+ to alpha s1-casein B and to a fragment alpha s1-CN3, obtained by cyanogen bromide cleavage, gave approximate pK values for the binding groups and suggest the possibility of a conformational change induced by varying the concentration of divalent cation.
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PMID:NMR studies of the phosphoserine regions of bovine alpha s1- and beta-casein. Assignment of 31P resonances to specific phosphoserines and cation binding studied by measurement of enhancement of 1H relaxation rate. 640 19

The primary structure of human beta-casein has been determined by automated Edman degradation of the intact protein and of peptides derived therefrom by hydrolysis with trypsin and by chemical cleavage with cyanogen bromide. For each form of this multiphosphorylated protein (0-5 P/molecule), phosphorylated sites at specific seryl and threonyl residues have been identified. These are located near the amino terminus, within the first 10 residues of this 212-amino acid molecule. Sequence comparison of human beta-casein with the bovine and ovine proteins reveals 50% identity and a 10-residue shifted alignment relationship. Locations of prolyl and charged residues are generally conserved for the three homologues. The sequence data indicate the existence of genetic polymorphism involving uncharged residues in human beta-casein.
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PMID:Human beta-casein. Amino acid sequence and identification of phosphorylation sites. 671 39

The expression of casein genes is under the control of several hormones of which the most important are prolactin, glucocorticoids and progesterone. In pseudopregnant or mid-pregnant rabbit having partially developped but inactive mammary gland, prolactin induces casein synthesis. The phenomenon is accompanied by an accumulation of casein mRNAs and by a stimulation of their translation. The accumulation of casein mRNAs results from an acceleration of the transcription of the corresponding genes and from a stabilization of the mRNAs. These prolactin effects are amplified by glucocorticoids which are not per se inducers and they are inhibited by progesterone. The essential action of prolactin and glucocorticoids can be obtained in cultured mammary explants and epithelial cells. This induction is accompanied by a transformation of the mammary cell in which are accumulated ribosomes and membranes involved in milk synthesis and exportation. This transformation is favoured by prolactin and inhibited by progesterone. Hence, the abundant milk secretion is triggered only after parturition when the predominence of progesterone is reversed in favour of prolactin. Prolactin incubated with mammary membranes promotes the formation of a factor capable of accelerating beta-casein gene transcription when added to isolated mammary nuclei. This factor is formed only by lactogen hormones and from prolactin receptor containing membranes. The information contained in the factor seems to be understood only by prolactin target genes. The generation of the factor can be provoked by anti-prolactin receptor antibodies and it is inhibited by tubulin binding drugs such as colchicine. The molecule exhibiting prolactin-like activity has a small molecular weight, it is thermostable and inactivated by trypsin. The stimulation of beta-casein gene transcription is abolished when the factor is incubated with nuclei in the presence of phosphatase inhibitors. These facts suggest that prolactin after its binding to its peripheral receptors triggers the release of a small peptide which migrates to nuclei where it activates the transcription of the prolactin target genes through a dephosphorylation of nuclear proteins. This small peptide is a good candidate to the prolactin intracellular relay.
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PMID:[Control of the expression of milk protein genes by prolactin, glucocorticoids and progesterone]. 676 97

The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immunoblotting with polyclonal antibodies against beta- and alpha s1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4.6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as gamma 3-casein (beta-casein(f108-209)) and alpha s1-PL1 (alpha s1-casein(f80-199). In both cases, a Lys-X bond was hydrolysed, indicating the action of a trypsin-like enzyme in beta- and alpha s1-casein hydrolysis during the ripening of this variety of hard pressed cheese.
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PMID:Gel electrophoresis and immunoblotting for the detection of casein proteolysis in cheese. 760 74


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