Sequences of Sixteen Phosphoserine Peptides from Ovalbumins of Eight Species

Henderson, J.Y.; Moir, Arthur J. G.; Fothergill, Linda A.; Fothergill, John E.

doi:10.1111/j.1432-1033.1981.tb05165.x

Cited by 41 publications

(33 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…USA 80 (1983) ule membranes in the parathyroid cell may be important in exocytosis, but this has not been investigated in the parathyroid previously. Examples of secreted proteins that are phosphorylated include casein (17), ovalbumin (18), myosin light chain (19), and a Mr 68,000 protein from peritoneal mast cells (20). Phosphorylation ofpro-ACTH/endorphin-derived peptides has also recently been described (21).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of parathyroid secretory protein.

Bhargava¹,

Russell²,

Sherwood³

1983

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The phosphorylation ofproteins released into the medium of bovine-parathyroid gland slices or isolated cells incubated with 32Pi has been investigated. The primary protein phosphorylated had a Mr of68,000 and coeluted with newly synthesized parathyroid secretory protein (PSP) on Bio-Gel chromatography and on polyacrylamide gel electrophoresis, Isoelectric focusing of double-labeled samples ([asSimethionine and 32P;) revealed comigration of the two radioactive markers at a pH of.4.6, which was similar to that of purified PSP. Phosphorylation of the Mr 68,000, protein was also-demonstrated in cell homogenates incubated with [7-32P] Methods. Fresh bovine parathyroid glands were obtained from the slaughterhouse. and kept on ice.. They were trimmed of fat and then slices were prepared with a Stadie-'vRiggs tissue slicer. The slices were -incubated with 3$P1 (100 ,ACi/ml) with and without [asS]methionine (10 p.Ci/ml) in a shaking water bath for 2-4 hr at 370C.in phosphate-free Krebs-Ringer bicarbonate-buffer (KRB buffer; 120 mM NaCl/4.75,mM KCl/1.2 mM MgCl2/0.5 or 2.5 mM CaCl2/25 mM NaHCO3, pH 7.4, containing 2 mg ofglucose per ml) in an atmosphere of95% 02/ 5% CO2. At the-end of the incubation period, the medium was separated from tissue by centrifugation at 650 x g for 10 min in -a refrigerated centrifuge. Medium was then centrifuged at 8,500 x g for 20 min and used for further studies. Isolated cells were prepared by the method of Brown et aL (12) and were incubated with 32Pj in phosphate-free KRB buffer. Cells and media were then processed for NaDodSO4/polyacrylamide gel electrophoresis or column chromatography on Bio-Gel A-1.5m (1.2 x 75 cm) in 0.15 M ammonium bicarbonate pH 7.8 buffer.After column chromatography, the radioactive peak fractions were pooled, -lyophilized, and analyzed further.For polyacrylamide gel electrophoresis, the proteins in the medium were precipitated by addition of an equal volume of 20% trichloroacetic acid at 40C. The medium was then washed with 10% and 5% trichloroacetic acid; extracted with ether, and dissolved in NaDodSO4 sample buffer [125 mM Tris-HCl, pH 6.8/1% NaDodSO4/5% (vol/vol) 2-mercaptoethanol/10% (vol/ vol) glycerol]. NaDodSO4/polyacrylamide gel electrophoresis was performed on 10% acrylamide slab gels with the discontinuous buffer system described by Maizel (13). Gel scanning was performed with a densitometer (E. C. Apparatus, St. Petersburg, FL). RESULTS When parathyroid gland slices or isolated cells were incubated for 120 min with 32Piand the medium proteins were precipitated and separated on polyacrylamide gel electrophoresis followed by radioautography, the dominant protein was a 68,000-dalton 32P-labeled protein which eluted in a position corresponding to that of highly purified bovine PSP (Fig. 1). Gel filtration (Bio-Gel A-1.5m) of 32P-labeled medium proteins revealed a peak ofradioactivity in the position where marker 125I-labeled PSP (125I-PSP) eluted (Fig. 2) analyzed by radioimmunoassay for bovine PSP -(5), immunoreactivity appeared only in the dominan...

show abstract

Section: Discussionmentioning

confidence: 99%

Phosphorylation of parathyroid secretory protein.

Bhargava¹,

Russell²,

Sherwood³

1983

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…, (317-319) and Ser-Glu-Glu (355-357). In these cases it is unlikely that the 'middle' residue has prevented phosphorylation since phosphoserine-344 is followed by an alanine (as it is in all eight ovalbumins) and also peptides from Chinese goose and Aylesbury duck ovalbumins are both phosphorylated at Ser-Met-Glu sequences [17].…”

Section: The Structure Of Ovalhuminmentioning

confidence: 99%

“…As in the case of the glycosylation site, thesc sites of post-synthetic modification are probably situated on the surface of the ovalbumin molecule. From a comparison of sixteen phosphoserine peptide sequences from ovalbumins from eight species it is notable that a glutamic acid residue always occurs two residues toward the C terminus from the phosphorylated aerine [17]. Caseins also have phosphorylated serines, and in aql casein and /j casein the phosphoserines are always followed in the n + 2 position by glutamic acid or another phosphoserine [42,43].…”

Section: The Structure Of Ovalhuminmentioning

confidence: 99%

See 1 more Smart Citation

The Complete Amino‐Acid Sequence of Hen Ovalbumin

Nisbet

Saundry

Moir

et al. 1981

European Journal of Biochemistry

Self Cite

451

245

View full text Add to dashboard Cite

The complete amino acid sequence of hen ovalbumin, comprising 385 residues, has been determined. The sequence was deduced from the 17 cyanogen bromide fragments and from peptides derived by digestion with a number of proteolytic enzymes. The molecular weight of the polypeptide chain of ovalbumin is 42699.Ovalbumin has four sites of postsynthetic modification ; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asn-292, and the two phosphorylated serines are at residues 68 and 344. The 'signal sequence' of ovalbumin is between residues 234 and 252. The heptapeptide released during the conversion of ovalbumin to plakalbumin by subtilisin digestion corresponds to residues 346 -352. The hen ovalbumin polymorphism characterised by an Asn-tAsp replacement results from a mutation at residue 31 1.The amino acid sequence of ovalbumin deduced from these amino acid sequence studies is in complete agreement with the sequence of mRNA determined by McReynolds et al. [Nature (Lond.) 273, 723 -728 (1978)].Ovalbumin is the most abundant protein in egg white, and is readily purified and crystallised in gram quantities [l].

show abstract

“…The CNBr fragment containing the reactive thiol group was eluted with several other fragments and required further purification by high-voltage paper electrophoresis at pH 2 [21].…”

Section: Cnbr Cleavage and Separation Qffragment5mentioning

confidence: 99%

A comparison of the structure and activity of cat and trout muscle pyruvate kinases

Harkins¹,

Nocton

Russell

et al. 1983

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Pyruvate kinase was purified from cat and trout muscle. The enzymes had similar amino acid compositions and subunit molecular weights. In contrast to the mammalian enzyme, the trout muscle pyruvate kinase was activated by fructose 1,6-bisphosphate. However, unlike the 1.-type pyruvate kinase from mammalian liver it was not phosphorylated by cyclic-AMP-dependent protein kinase. The purified enzyme from cat muscle was carboxymethylated with iod0[2-'~C]acetic acid under conditions that led to the preferential labelling of one especially reactive thiol group. The labelled enzyme was cleaved with CNBr, and the radioactive fragment purified. Amino acid sequence analysis of the reactive-thiol-containing fragment from cat muscle pyruvate kinase showed it had the following sequence : Ile-Gly-Arg-['4C]CmCys-Asn-Arg-Ala-Gly-Lys-Pro-Va~-Ile-CmCys-Ala-Thr-Gln-Hse. The corresponding peptide from trout pyruvate kinase had only one difference in its amino acid composition and the sequence around the reactive thiol was identical.Pyruvate kinase is a key regulatory enzyme found in all cells and tissues during glycolysis, and catalyses the following reaction :The properties of the enzyme have been reviewed by Boyer [I] and Kayne 121. The reaction is virtually irreversible and requires a bivalent cation, such as Mg2+ or Mn2+, and a monovalent cation, normally K+. The enzyme is a tetramer with identical subunits of M , x 60000. The regulation of the activity of pyruvate kinase is important in the control of glycolysis, especially in tissues capable of gluconeogenesis. Isolation of the enzyme from different tissues has led to the identification of three major classes of isoenzymes. The L-type enzyme (first isolated from mammalian liver) shows a sigmoidal dependence of activity on P-pyruvate concentration, and is inhibited by gluconeogenic amino acids such as alanine. The affinity for Ppyruvate is enhanced by the addition of the activator, fructose 1,6-bisphosphate. The L-type enzyme is also allosterically activated by H+, and inhibited by ATP. In contrast, the M-type enzyme (first isolated from mammalian muscle) has been considered not to possess allosteric properties, although conditions have been found under which the muscle enzyme displays kinetic properties typical of a regulatory enzyme [3]. The A-type pyruvate kinase has properties intermediate between those of the M-type and L-type enzymes.M-type pyruvate kinase has been studied extensively; the X-ray crystallographic structure of the enzyme from cat muscle is known at a resolution of 0.26 nm [4]. It has been shown by low-resolution studies that each subunit has one ATPIPpyruvate/Mn2 + binding site and a secondary (non-catalytic)ADP binding site 151. Much less is known of the structure of Ltype pyruvate kinase. The liver enzyme has been shown to be phosphorylated on a specific serine residue by incubation with [32P]ATP and cyclic-AMP-dependent protein kinase, with a concomitant decrease in activity [6]. These studies show that the inhibitory effect of phosphorylation is coun...

show abstract

Sequences of Sixteen Phosphoserine Peptides from Ovalbumins of Eight Species

Cited by 41 publications

References 31 publications

Phosphorylation of parathyroid secretory protein.

Phosphorylation of parathyroid secretory protein.

The Complete Amino‐Acid Sequence of Hen Ovalbumin

A comparison of the structure and activity of cat and trout muscle pyruvate kinases

Contact Info

Product

Resources

About