Stable pattern formation and determination of molecular size by pore-limit electrophoresis

Slater, Grant G.

doi:10.1021/ac60277a003

Cited by 102 publications

(30 citation statements)

References 10 publications

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“…Native polyacrylamide gel electrophoresis was conducted in 5 -25% polyacrylamide gradient gels as described by Slater [18] using the buffer system of Davis [19]. After electrophoresis, gels were stained for protein using Coomassie brilliant blue R 250, for glycoprotein using periodic acid/ Schiff reagent and for lipoprotein using Sudan black B. Molecular mass standards used were: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), albumin dimer (134 kDa) and monomer (67 kDa).…”

Section: Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Yolk protein in leech

Baert

Britel

Slomianny

et al. 1991

European Journal of Biochemistry

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Tlzeromyzon tessulatum vitellin was identified as a lipoglycoprotein of 490 kDa. The insolubility of this molecule in low-ionic-strength media was used to extract it from the ovaries. Antiserum prepared against vitellin was shown to react with a coelomic fluid component of 520 kDa. This vitellin precursor, or vitellogenin, was purified by gel permeation and ion-exchange column chromatography. These two lipoglycoproteins were characterized by amino acid, carbohydrate and lipid analysis and subunit composition. In spite of differences in terms of native molecular mass, solubility and isoelectric point, the lipoglycoproteins isolated from the coelomic fluid and the ovary were similar in their subunit components (a single polypeptide of 165 kDa) and in their amino acid and carbohydrate compositions. However, vitellogenin was found to be more highly lipidated (31.8% by mass) than vitellin (24% by mass) and lipid analysis indicated a higher amount of sterols and phospholipids in vitellogenin. From these data, we conclude that vitellogenin and vitellin are probably dimers of two identical subunit polypeptides plus lipid and that, after vitellogenin is sequestered in the oocyte, part of its lipid component is stripped from the molecule to give vitellin. Furthermore, electrophoretic analysis seems to indicate that vitellogenin synthesis and secretion is initiated following the third and last blood meal of the animal but that vitellogenin significantly accumulates in the coelomic fluid before being incorporated in the oocytes suggesting a complex mode of vitellogenesis regulation.The production of egg yolk involves a massive synthesis of specific proteins which is under hormonal control. These proteins, termed vitellogenins, are generally synthesized externally to the site of accumulation. Depending upon the species, vitellogenins are secreted in the bloodstream or in the hemolymph, taken up by the developing oocytes and deposited as vitellins, the major yolk proteins [l, 21. Thus, in addition to its physiological importance for reproduction, vitellogenesis also provides a good system for the study of molecular mechanisms and hormonal regulation of egg maturation.Yolk proteins have been extensively investigated in oviparous vertebrates [I] and in insects [3, 41. In annelids, oocyte vitellin has been purified from nereid polychaetes [5, 61 and it has been shown that oocyte differentiation depends upon the incorporation of vitellogenin [7, 81. On the other hand, little is known for the two groups of clitellates: oligochaetes [9, 101 and hirudinae [I I]. In clitellates, eggs are generally of a small size and laid within a cocoon secreted by the clitellum and abundantly provided with reserve substances from which the embryo will develop, therefore making an accumulation of

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Section: Polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Yolk protein in leech

Baert

Britel

Slomianny

et al. 1991

European Journal of Biochemistry

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“…Native polyacrylamide gel electrophoresis was conducted in 5 -25% polyacrylamide gradient gels as described by Slater [14] using the buffer system of Davis [15]. After electrophoresis, proteins were stained with Coomassie blue and their relative molecular masses were determined using marker proteins (Pharmacia).…”

Section: Polyacrylumide Gel Electrophoresismentioning

confidence: 99%

A 15000‐M_r protein proteolytically derived from vitellogenin within oocyte of Perinereis cultrifera (polychaete annelid)

Rahuel

London

Vignal

et al. 1988

European Journal of Biochemistry

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It has been shown previously in our laboratory that, in Perinereis cultrifera, the four mature vitellin subunits (Mr 98000, 22000, 20000, 16000) are proteolytically derived within the oocyte from a single extraoocytic precursor, vitellogenin, with an apparent Mr (176000) higher by 20000 than the sum of the Mr of the four end products. In this report, it is shown that a 15000‐Mr protein, designated as P15, not only accumulates in maturing oocytes but also originates from outside these cells similarly to vitellin. Moreover in vivo labelling experiments indicate that the appearance of P15 occurs after vitellogenin enters the oocyte, concurrently with the appearance of the lower‐Mr fragments characteristic of vitellin. From these data, it is concluded that P15 most likely represents a vitellogenin‐derived protein which is generated within the oocytes during the transformation of vitellogenin into vitellin. This conclusion is further supported by the additional finding that P15 immunologically cross‐reacts with vitellogenin but not with mature vitellin. P15 has been purified to homogeneity from the soluble protein fraction of submature oocytes and partially characterized. The 15000‐Mr protein exists in a monomeric form with a pI of about 7.7. Unlike vitellin, P15 does not contain significant amounts of lipid or carbohydrate and has a low absorbance at 280 nm. The amino acid composition of the purified protein is also presented.

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“…For screening of the AGT gene, an oligonucleotide mixture probe (a mixture of 128 sequences [5Ј-ATGGAYTAYACNA ARTAYYT-3Ј]) was synthesized based on the N-terminal amino acid sequence (underlined) of the enzyme as determined in this study (MDYTKYLAGRANWIKG). The probe was labeled with [␥- 32 P]ATP by the use of T4 polynucleotide kinase and Megalabel (Takara Biochemicals, Kyoto, Japan), purified through a ProbeQuant G-50 Micro column (Amersham Bioscience, Tokyo, Japan), and used as a specific probe for colony and Southern hybridizations. To obtain a clone containing the AGT gene, we prepared T. litoralis chromosomal DNA as previously described (22), digested it with several restriction enzymes, and then separated the fragments by 0.8% agarose gel electrophoresis.…”

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confidence: 99%