scholarly journals Liberation of tryptic fragments from caseinomacropeptide of bovine κ-casein involved in platelet function. Kinetic study

1990 ◽  
Vol 271 (1) ◽  
pp. 247-252 ◽  
Author(s):  
J Léonil ◽  
D Mollé

Carbohydrate-free caseinomacropeptide (CMP) was purified from rennet-hydrolysed caseinate by trichloroacetic acid precipitation and DEAE-TSK Fractogel-650 ion-exchange chromatography. To study the liberation of 106-112, 106-116 and 113-116 fragments from carbohydrate-free CMP involved in platelet function, a quantitative study was made on the rate of hydrolysis of the three peptidic bonds that are susceptible to the action of trypsin. Data were obtained from reverse-phase (Ultrabase column) and cationic-exchange (Mono S column) h.p.l.c. On the basis of the disappearance of substrate, kcat. and Km were respectively 3.95 s-1 and 0.2 mM. The two 111-112 and 112-113 bonds were split according to similar kinetic parameters (kcat. = 1.97 s-1, Km = 0.2 mM) and much faster than the 116-117 bond. The difference in susceptibility of the bonds can probably be attributed to the nature of residues flanking the primary proteolytic sites rather than to their accessibility to the proteinase. On the basis of our results the 106-116 fragment cannot be formed.

1973 ◽  
Vol 45 (6) ◽  
pp. 849-858 ◽  
Author(s):  
D. J. Boullin ◽  
R. F. Crampton ◽  
Christine E. Heading ◽  
D. Pelling

1. The intestinal absorption of carnosine, glycylglycine, glycyl-d-phenylalanine, glycyl-l-phenylalanine, glycyl-l-proline and l-prolylglycine were investigated after intraluminal injection of dipeptide into anaesthetized rats. 2. With all six dipeptides, the intact substance was detected by ion-exchange chromatography in blood samples taken from the superior mesenteric vein. 3. The rate of hydrolysis of the dipeptides in tissue homogenates was measured in vitro. 4. The relative rates of hydrolysis varied by a factor of 300; there was an apparent inverse relationship between rate of hydrolysis and detection of intact peptide. 5. Peptide absorption was accompanied by increases in venous concentrations of the component amino acids, which appeared in proportions appropriate to the view that peptide absorption preceded hydrolysis. 6. It is suggested that slowly hydrolysed dipeptides may pass intact through the intestine wall under physiological conditions.


1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.


2018 ◽  
Vol 20 (1) ◽  
pp. 56-60 ◽  
Author(s):  
Tsutomu Arakawa

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.


1979 ◽  
Vol 177 (3) ◽  
pp. 967-973 ◽  
Author(s):  
G P Margison ◽  
J M Margison ◽  
R Montesano

1. Syrian golden hamster liver ribosomal RNA was isolated up to 96 h after administration of [14C]dimethylnitrosamine at 25 mg/kg or 2.5 mg/kg body weight. 2. The chemical alkyation products, 7-methylguanine, 3-methylcytosine, O6-methylguanosine and 1-methyladenosine, were measured after acidic or enzymic hydrolysis of the RNA to bases or mononucleosides followed by ion-exchange chromatography. 3. Between 7 and 96 h, the relative amounts of alkylation products did not change with time even though the absolute amounts fell by approx. 80% and 51% after the high and low doses respectively. 4. The results suggest that base specific excision repair does not exist for RNA alkylation products in this experimental system.


1954 ◽  
Vol 44 (01) ◽  
pp. 7-44 ◽  
Author(s):  
W.E. Boyd

Summary1. A method is described for investigating the possible action of microdoses of mercuric chloride on the hydrolysis of soluble starch with malt diastase.2. The microdoses of the mercuric chloride used in the latest crucial series carried out in1946, 1948, and 1952, were what are termed “high potencies” made in accordance with the pharmaceutical method of preparation of drugs ordinarily used in the practice of homœotherapy.3. These microdoses were prepared by separate stages of dilution, the solution at each stage being subjected to mechanical shock. The solutions were, theoretically, “dilutions” of the order of 1 in 10−61 and on present physical theory would not contain any molecules of the original mercuric chloride.4. The difference in rate of hydrolysis between flasks containing starch, diastase, and distilledwater (controls) and flasks containing starch, diastase and microdoses of mercuric chloride (tests) were compared colorimetrically by the Spekker absorptiometer, and the frequencies of the differences statistically analysed, as the results obtained showed biological scatter. More than 500 such comparisons were carried out. The differences of means were examined by the Fisher “t” test, the variances tested and Cochrane and Cox's test applied where indicated. All the series gave a highly significant difference in the rate of hydrolysis between controls and tests, the microdoses stimulating the process. Statistically the significance is shown by the fact that a probability of <0·001 was obtained independently in each of the three years 1946, 1948 and 1952. The control results gave an approximately normal distribution.5. The distribution, control methods, and accessory control procedures were considered toexclude, as a cause of the effects, adsorption of the original drug and the presence of extraneous contaminants by chance solely in test flasks. The only difference between control and microdose flasks was the addition of microdose, the distilled water being common to both controls and tests.6. It was concluded that a factor, unidentified, derived from the mercuric chloride used, waspresent in solutions prepared by serial dilution with mechanical shock which could affect the distilled water diluent, that this change was transferable to subsequent “ultramolecular” stages of “dilution”, and that this factor was the source of the activity in the microdose solutions producing the acceleration of the rate of hydrolysis.7. In an addendum there is described recent biological work which is also providing evidence of the presence of an active selective factor in “high potencies” derived from Strophanthus sarmentosus by the same methods of dilution with mechanical shock.


2006 ◽  
Vol 72 (5) ◽  
pp. 3383-3389 ◽  
Author(s):  
Takeshi Zendo ◽  
Shoko Koga ◽  
Yasushi Shigeri ◽  
Jiro Nakayama ◽  
Kenji Sonomoto

ABSTRACT A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, α and β, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qα plus Qβ) and lactococcin G (Gα plus Gβ) clarified that hybrid combinations (Qα plus Gβ and Gα plus Qβ) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qα plus Gβ was 32 times higher than that of Qα plus Qβ, suggesting that the difference in β peptides was important for the intensity of antibacterial activity.


1977 ◽  
Vol 55 (4) ◽  
pp. 315-324 ◽  
Author(s):  
Peter Hechtman

Separation of the hexosaminidase A (EC 3.2.1.52) and B isozymes of human liver by ion-exchange chromatography results in recovery of greater than 80% of the activity in crude extracts when synthetic substrates are used to monitor enzyme activity. Only 15% of hexosaminidase activity toward the N-acetylgalactosaminyl (N-acetylneuraminyl) galactosyl glucosylceramide (Gm2 ganglioside) substrate is recovered and all of this activity is associated with the hexosaminidase A fraction.The low level of Gm2 ganglioside hydrolase activity in the hexosaminidase A fraction could be enhanced by coincubation with column fractions which contain hexosaminidase B. The activating factor, which has been partially purified by gel filtration, is a heat-stable protein with a molecular weight of 36 000 and is without enzyme activity toward hexosaminidase substrates.Highly purified hexosaminidase A or crude hexosaminidase A recovered after gel filtration on Sephadex G-100 has no Gm2 ganglioside hydrolase activity. The Gm2 ganglioside hydrolase activity of these hexosaminidase A preparations can be completely restored by addition of activating factor. The activating factor does not affect the rate of hydrolysis of synthetic substrate or asialo Gm2 ganglioside catalyzed by hexosaminidase A.


2018 ◽  
Vol 6 (2) ◽  
pp. 69
Author(s):  
Indah Rahmi Sari ◽  
Ade Ayu Oksari ◽  
Irma Kresnawaty

Separation of Amino acid from Liquid waste of Oil palm Factory with Ion Exchange Chromatography Research on Separation of Amino Acid Liquid Waste mills with Ion Exchange Chromatography was carried out from October to November 2015. The results of  hydrolysis of 6 N HCl results showed  that the highest absorbance reading was obtained at a concentration of eluent of 0,2 and 0,6 M NaCl, while the results of the protease enzyme hydrolysis the highest absorbance reading at NaCl eluent 0,2 and 1 M. There was no significant difference in the results of separation by ion exchange chromatography, showed that the concentration of NaCl eluent is not very influential, so for subsequent analysis used only one concentration of the eluent. Results of linear regression obtained was equal to 0,9946, these results indicate that the series standard amino acid lysine has a value that is linear as it approaches 1. The amino acid levels obtained on the sample results LCPKS hydrolysis with 6 N HCl which was about 0 to 8.82 ppm and samples of the protease enzyme hydrolysis of about 0 to 4.31 ppm. Amino acid levels obtained were still far from the expected.Keywords: Amino Acid, Oil Palm, Liquid Waste, Ion Exchange Chromatography ABSTRAKPenelitian mengenai Pemisahan Asam Amino dari Limbah Cair Pabrik Kelapa Sawit dengan Kromatografi Penukar Ion telah dilaksanakan dari bulan Oktober sampai November 2015. Hasil hidrolisis HCl 6 N menunjukkan bahwa pembacaan absorbansi tertinggi diperoleh pada konsentrasi eluen NaCl 0,2 dan 0,6 M, sedangkan hasil hidrolisis enzim protease pembacaan absorbansi tertinggi pada eluen NaCl 0,2 dan 1 M. Tidak ada perbedaan yang signifikan pada hasil pemisahan dengan kromatografi penukar ion ini, menunjukkan bahwa konsentrasi eluen NaCl tidak terlalu berpengaruh, sehingga untuk analisis selanjutnya digunakan hanya satu konsentrasi eluen. Hasil regresi linear yang diperoleh yaitu sebesar 0,9946, hasil tersebut menunjukkan bahwa deret standar asam amino lisin mempunyai nilai yang linear karena mendekati 1. Kadar asam amino yang diperoleh pada sampel hasil hidrolisis LCPKS dengan HCl 6N yaitu sekitar 0 – 8,82 ppm dan sampel hasil hidrolisis enzim protease sekitar 0 – 4,31 ppm. Kadar asam amino yang diperoleh masih jauh dari yang diharapkan.Kata Kunci: Asam Amino, Minyak Kelapa Sawit, Limbah Cair, Kromatografi Penukar Ion


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