scholarly journals Primary structure of the ovine pituitary follitropin α-subunit

1981 ◽  
Vol 197 (3) ◽  
pp. 535-539 ◽  
Author(s):  
M R Sairam
Keyword(s):  
Sequence Analysis ◽  
Primary Structure ◽  
Alpha Subunit ◽  
Partial Sequence ◽  
Tryptic Peptides ◽  
Α Subunit ◽  
Alpha Subunits ◽  
Entire Structure

All the tryptic peptides of reduced and aminoethylated alpha-subunit of ovine pituitary follitropin were isolated. From their their composition and partial sequence analysis of the N- and C-termini of the oxidized protein and reliance on homology with the sequence of lutropin alpha-subunit, an entire structure for the follitropin alpha-subunit has been proposed. The structure of the alpha-subunits from the two ovine hormones are identical.

scholarly journals Proteolytic processing of the α-subunit of rat endopeptidase-24.18 by furin

1995 ◽  
Vol 309 (2) ◽  
pp. 683-688 ◽  
Author(s):  
P E Milhiet ◽  
S Chevallier ◽  
D Corbeil ◽  
N G Seidah ◽  
P Crine ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Consensus Sequence ◽  
Proteolytic Processing ◽  
Alpha Subunit ◽  
Structural Domain ◽  
Α Subunit ◽  
Alpha Subunits ◽  
Golgi Compartment ◽  
Membrane Bound

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.

scholarly journals Expression of wild-type and mutated rabbit osteopontin in Escherichia coli, and their effects on adhesion and migration of P388D1 cells

1995 ◽  
Vol 307 (1) ◽  
pp. 257-265 ◽  
Author(s):  
K Nasu ◽  
T Ishida ◽  
M Setoguchi ◽  
Y Higuchi ◽  
S Akizuki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluid Phase ◽  
Intradermal Injection ◽  
Polymorphonuclear Leucocyte ◽  
Alpha Subunit ◽  
Wild Type ◽  
Alpha Subunits ◽  
Leucocyte Migration ◽  
And Migration ◽  
Chemotactic Effect

Recombinant wild-type rabbit osteopontin (rOP) and the protein with an aspartate-to-glutamate transposition induced by a point mutation in the rabbit OP cDNA within the Gly-Arg-Gly-Asp-Ser (GRGDS) sequence were expressed in Escherichia coli and purified to homogeneity. P388D1 cells bound rOP in a saturable manner. rOP induced adhesion and haptotaxis of P388D1 cells, whereas mutated rabbit OP (rOPmut) did not. Anti-rOP IgG F(ab′)2 and synthetic GRGDS peptide inhibited rOP-mediated adhesion and haptotaxis of P388D1 cells. Fibronectin (FN)-mediated adhesion of P388D1 cells was markedly inhibited in the presence of fluid-phase rOP. Adhesion of P388D1 cells to rOP was significantly inhibited by anti-[alpha-subunits of VLA4 (alpha 4) and VLA5 (alpha 5)] monoclonal antibodies (mAbs), but not by anti-[alpha-subunit of vitronectin (VN) receptor (alpha V) or Mac-1 (alpha M)] mAb. Adhesion of P388D1 cells to FN and VN was significantly inhibited by anti-alpha V mAb but not anti-alpha 4, -alpha 5 or -alpha M mAb. Haptotaxis of P388D1 cells to rOP was significantly inhibited by anti-alpha V mAb, but not by anti-alpha 4, -alpha 5 and alpha M mAbs, whereas that to FN showed no inhibition with all three mAbs. Haptotaxis of P388D1 cells to VN was significantly inhibited by anti-alpha 5 and -alpha V mAbs but not by anti-alpha 4 and -alpha M mAbs. Similar features of inhibition of adhesion and haptotaxis of P388D1 cells to human OP were observed by mAbs. rOP had no chemotactic effect on P388D1 cells. Significant polymorphonuclear leucocyte migration was observed 3-12 h after intradermal injection of rOP into rabbits.

scholarly journals Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin

1980 ◽  
Vol 192 (2) ◽  
pp. 469-481 ◽  
Author(s):  
W A Hughes ◽  
R W Brownsey ◽  
R M Denton
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Subcellular Fractionation ◽  
Alpha Subunit ◽  
Fat Cells ◽  
Phosphorylated Proteins ◽  
Alpha Subunits ◽  
Dehydrogenase Complex

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35–45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.

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