Production of sialic acid rich glycopeptide from bovine κ-casein glycomacropeptide by hydrolyzing with papain

AbstractBovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide having many biological activities. The study described in this research communication was undertaken to determine whether sialic acid rich glycopeptide can be produced from GMP by proteinase treatment. A sample of GMP was hydrolyzed with papain, and the obtained hydrolysate was chromatographed on a column of diethylaminoethyl-Sephacel to obtain a glycopeptide fraction (GPF). This product accounted for average 48.1% dry weight of GMP or 81.1% total recovered sialic acid from GMP. The content of sialic acid (expressed as % dry weight) was 1.7 times higher in GPF (22.6) than in unhydrolyzed GMP (13.4). Major differences in amino acid composition between GPF and GMP were found in the contents (mol%) of: lysine (<1 and 4.5, respectively), serine (20.3 and 10.3, approximately twice higher in GPF), asparagine/aspartic acid and isoleucine. The contents of the last two amino acids were approximately twice lower in GPF. On gel filtration chromatography with Sephacryl S-100, GMP was eluted as a single peak with elution volume similar to that of dimeric β-lactoglobulin (36.6 kDa) whereas GPF was eluted in two peaks both with elution volumes greater than that of α-lactalbumin (14.2 kDa). These peak fractions containing high (fraction I) and low (fraction II) molecular size glycopeptides gave different sialic acid to peptide ratio, which was 1.7 times higher in fraction I than in fraction II. Results of size exclusion HPLC on Superdex-75 were consistent with those of gel filtlation chromatography. On cellulose acetate electrophoresis, the mobility of GPF relative to that of GMP as 1.0 was found to average 1.2, suggesting a higher negative charge density in GPF than in GMP. It was concluded that papain digestion of GMP is an efficient method to produce glycopeptide with high sialic acid content.

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Correction of the Delayed Fibrin Aggregation of Fetal Fibrinogen by Partial Removal of Sialic Acid

10.1055/s-0039-1684458 ◽

1979 ◽

Author(s):

D.K. Galanakis ◽

M.W. Mosesson

Keyword(s):

Sialic Acid ◽

Acid Content ◽

Acid Value ◽

Full Term ◽

Thrombin Time ◽

Sialic Acid Content ◽

Vibrio Cholera ◽

The Mean ◽

Fraction I ◽

Free Sialic Acid

Human umbilical oord fibrinogen characteristically displays delayed fibrin aggregation under conditions of relatively high ionic strength. This delay is greater in fibrinogen obtained from premature (p) (24–35 weeks gestation) infants, as compared with that from full term (FT) infants. We compared the sialic acid content of (fraction I-2) fetal (P and FT) with adult (A) fibrinogen, obtained from pooled plasma. The mean sialic acid μg/100mg protein) values were: P, 818 (±135 SD, range 621–1030); FT, 720 (±212, range 505–1280); A, 626 (±110, range 487-806). One P fibrinogen preparation (thrombin time 22.6 seconds) was partially desialated (resulting sialic acid value 490) by incubation with Vibrio cholera neuraminidase, dialyzed , and precipitated with ethanol to remove free sialic acid. The thrombin time of the resulting preparation was 15.4 (range 15.2–15.8), compared to 16.1 (range 15.5–16.4) for untreated A fibrinogen. The results suggest that the delayed fibrin aggregation of fetal fibrinogen is attributable to its relatively high sialic acid content. Moreover, the intermediate sialic acid (and thrombin time) values of FT (as compared to those of p) fibrinogen intimate the presence of a mixture of adult and fetal fibrinogen in full term cord blood.

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The influence of sialic acid residues on the ability of glycoproteins toi nteract electrostatically with chondromucoprotein

Biochemical Journal ◽

10.1042/bj0970333 ◽

1965 ◽

Vol 97 (2) ◽

pp. 333-339 ◽

Cited By ~ 4

Author(s):

AJ Anderson

Keyword(s):

Sialic Acid ◽

Complex Formation ◽

Acid Hydrolysis ◽

Acid Content ◽

Biological Activities ◽

Carbohydrate Composition ◽

Mild Acid Hydrolysis ◽

Sialic Acid Content ◽

Neuraminidase Treatment ◽

Mild Acid

1. Although glycoproteins with less than 1% of sialic acid (fibrinogen, lipoproteins, gamma-globulins) interact electrostatically with chondromucoprotein to form insoluble complexes, interaction with glycoproteins containing larger amounts of sialic acid (orosomucoid, urine glycoprotein, seromucoid, fraction VI) was electrostatically impossible. Reasons for this are discussed. 2. The latter glycoproteins interacted with chondromucoprotein after mild acid hydrolysis or neuraminidase treatment, complex-formation being inversely related to their sialic acid content. 3. Complex-formation with sialic acid-deficient orosomucoid was maximum at pH3.6 and negligible above its isoelectric point of pH5, and was inhibited by Ca(2+) ions and EDTA. 4. These results are discussed in relation to the carbohydrate composition and biological activities of euglobulin fractions, and of complexes formed by adding chondromucoprotein to abnormal plasmas which may contain sialic acid-deficient glycoproteins owing to faulty carbohydrate metabolism.

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THE RELATIONSHIP OF THE INFLUENZA VIRUS INHIBITORY ACTIVITY OF GLYCOPROTEINS TO THEIR MOLECULAR SIZE AND SIALIC ACID CONTENT

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.64.2.634 ◽

1969 ◽

Vol 64 (2) ◽

pp. 634-641 ◽

Cited By ~ 27

Author(s):

G. F. Springer ◽

H. G. Schwick ◽

M. A. Fletcher

Keyword(s):

Influenza Virus ◽

Sialic Acid ◽

Inhibitory Activity ◽

Acid Content ◽

Molecular Size ◽

Sialic Acid Content ◽

Relationship Of ◽

The Relationship

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Immunochemical and biological characterization of calcitonin originating from transplanted medullary thyroid carcinoma in rats

Acta Endocrinologica ◽

10.1530/acta.0.0990397 ◽

1982 ◽

Vol 99 (3) ◽

pp. 397-403 ◽

Cited By ~ 1

Author(s):

Lisbeth Myhre ◽

Arne Ekeland ◽

Kaare M. Gautvik

Keyword(s):

Biological Activities ◽

Gel Filtration ◽

Molecular Size ◽

Serum Levels ◽

Biologically Active ◽

Subcutaneous Injections ◽

Medullary Thyroid ◽

Biological Potency ◽

Medullary Thyroid Carcinomas

Abstract. The immunoreactive and biological activities of calcitonin (CT) produced by transplanted rat medullary thyroid carcinomas (MCT) have been studied. Immunoreactive CT (iCT) in serum and in MCT tissues of rats carrying tumours of generations 5–8 was characterized by gel filtration on Sephadex G-100 followed by radioimmunological measurements using region specific antiserum. The hypocalcaemic effect of sera and tumour extracts was tested in a rat bioassay. Rats with transplanted MCT of the 5th and 6th generations had mainly (70–84%) circulating iCT species of molecular size comparable to intact hormone. However, in rats with tumours of the 7th and 8th generations corresponding circulating iCT forms comprised less than 52% of total immunoreactivity while 32–38% eluted earlier. In conparison, iCT corresponding to intact hormone represented 30–50% of total immunoreactivity in the tumour extracts and no differences were observed between the generations. Subcutaneous injections of sera from MCT rats and of tumour extracts reduced the serum levels of ionized calcium in test rats. The sera containing mainly intact iCT showed the strongest biological potency. We conclude that rat MCT transplanted under the kidney capsule is able to secrete biologically active CT. However, the heterogeneity of circulating iCT increases in rats with transplanted tumours of older generations, approaching the heterogeneity of stored hormone in the gland.

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STUDIES ON HUMAN CHORIONIC GONADOTROPHIN

Acta Endocrinologica ◽

10.1530/acta.0.0590089 ◽

1968 ◽

Vol 59 (1) ◽

pp. 89-104 ◽

Cited By ~ 29

Author(s):

H. van Hell ◽

R. Matthijsen ◽

J. D. H. Homan

Keyword(s):

Sialic Acid ◽

Calcium Ions ◽

Gel Electrophoresis ◽

Gel Filtration ◽

Chorionic Gonadotrophin ◽

Human Chorionic Gonadotrophin ◽

Starch Gel Electrophoresis ◽

Sialic Acid Content ◽

Starch Gel ◽

Biological Potency

ABSTRACT Highly purified preparations of Human Chorionic Gonadotrophin (HCG), with potencies up to 18 800 IU/mg, were obtained from material containing about 1500 IU/mg, by using a fractionation with ethanol, CMSephadex chromatography and gel filtration with Sephadex G-100. Starch gel electrophoresis revealed that the most potent preparation still contained three components, while certain other fractions with much lower potencies were found to be homogeneous. Evidence is presented for the presence of various HCG components differing from each other in biological potency, electrophoretic mobility and sialic acid content. Within this material, components with higher electroporetic mobilities and higher sialic acid contents showed higher biological potencies. The molecular weight estimated in the absence of calcium ions was 62 000 ± 1000.

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Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody

Biochemical Journal ◽

10.1042/bj2850839 ◽

1992 ◽

Vol 285 (3) ◽

pp. 839-845 ◽

Cited By ~ 98

Author(s):

T P Patel ◽

R B Parekh ◽

B J Moellering ◽

C P Prior

Keyword(s):

Sialic Acid ◽

Profile Analysis ◽

Protein A ◽

Gel Filtration ◽

Culture Method ◽

Exchange Chromatography ◽

Glycosylation Pattern ◽

Culture Methods ◽

Sialic Acid Content ◽

Serum Free

A monoclonal IgG-1 was produced by culture of a murine hybridoma (3.8.6) by three different methods, namely culture in ascites, in serum-free media and in serum-supplemented media. IgG-1 was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) from each medium by ion-exchange chromatography and h.p.l.c. Protein A chromatography. Oligosaccharides were released from each IgG-1 preparation by hydrazinolysis and radiolabelled by reduction with alkaline sodium borotritide, and ‘profile’ analysis of the radiolabelled oligosaccharide alditols was performed by a combination of paper electrophoresis and gel-filtration chromatography. This analysis indicated clear and reproducible differences in the glycosylation patterns of the three IgG-1 preparations. Sequential exoglycosidase analysis of individual oligosaccharides derived from each IgG-1 preparation was used to define these differences. Ascites-derived material differed from serum-free-culture-derived material only with respect to the content of sialic acid. IgG-1 derived from culture in serum-containing media had an intermediate sialic acid content and a lower incidence of outer-arm galactosylation than the other two preparations. These differences in glycosylation could not be induced in any IgG-1 preparation by incubating purified IgG-1 with ascites or culture medium. It is concluded that the glycosylation pattern of a secreted monoclonal IgG is dependent on the culture method employed to obtain it.

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Peptide nature of two mosquito natriuretic factors

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1986.250.3.r328 ◽

1986 ◽

Vol 250 (3) ◽

pp. R328-R332 ◽

Cited By ~ 2

Author(s):

D. H. Petzel ◽

H. H. Hagedorn ◽

K. W. Beyenbach

Keyword(s):

Biological Activities ◽

Biological Effects ◽

Gel Filtration ◽

Fluid Secretion ◽

Malpighian Tubules ◽

Peptide Bonds ◽

Polar Low ◽

Transepithelial Voltage ◽

Fraction I

High-pressure liquid chromatography (HPLC) of saline extracts of Aedes aegypti heads yields three fractions (from a total of 108) that affect transepithelial voltage and/or fluid secretion in isolated Aedes Malpighian tubules. In this study we investigated the physical and chemical nature of the active materials in these fractions. Gel-filtration chromatography revealed that the molecular weights of the three fractions were between 1,900 and 2,700. To test their thermostability the fractions were repeatedly frozen and thawed over a period of 110 days without loss of biological activity. Boiling at 100 degrees C for 5 min failed to significantly reduce their biological effects in isolated Malpighian tubules. In contrast, treatment with the proteolytic enzyme mixture, pronase, destroyed activity in all three. Fraction I no longer depolarized the transepithelial voltage of in vitro perfused Malpighian tubules, and fractions II and III completely lost their ability to stimulate fluid secretion and to affect transepithelial voltage. We conclude that our HPLC isolation yields a heterogeneous group of three polar low-molecular weight peptides. Expression of their biological activities in Malpighian tubules depends on intact peptide bonds.

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The influence of fluoride administration on the structure of proteoglycans in the developing rat incisor

Biochemical Journal ◽

10.1042/bj1900263 ◽

1980 ◽

Vol 190 (2) ◽

pp. 263-272 ◽

Cited By ~ 30

Author(s):

J W Smalley ◽

G Embery

Keyword(s):

Intraperitoneal Administration ◽

Gel Filtration ◽

Deae Cellulose ◽

Molecular Size ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Rat Incisor ◽

Cellulose Acetate Electrophoresis ◽

Sulphate Proteoglycan ◽

Developing Rat

1. 35S-labelled chondroitin 4-sulphate proteoglycan was isolated from the mineralized elements of the developing incisor teeth of Harvard rats receiving intraperitoneal administration of Na235SO4. 2. The chondroitin 4-sulphate proteoglycan underwent a decrease in molecular size in fluorotic teeth as judged by gel filtration on Sepharose 2B. 3. When examined by anion-exchange chromatography on DEAE cellulose-52, the proteoglycan from fluorotic teeth resolved into four peaks in comparison with the material from non-fluorotic teeth, which exhibited only a single major peak. 4. Both the single peak from non-fluoridated teeth and the four peaks from the fluorotic teeth were further resolved on cellulose acetate electrophoresis. 5. Isolated chondroitin 4-sulphate chains obtained from fluorotic teeth also were of smaller molecular size as judged by gel filtration on Sephadex G-150. 6. Some possible influences of fluoride on the metabolism of these connective-tissue components in the developing rat incisor are discussed.

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