scholarly journals Preparation and characterization of novel substrates of insulin proteinase (EC 3.4.99.45)

1994 ◽  
Vol 302 (3) ◽  
pp. 907-911 ◽  
Author(s):  
R C Werlen ◽  
R E Offord ◽  
K Rose
Keyword(s):  
Cytochrome C ◽  
Cleavage Sites ◽  
Rat Skeletal Muscle ◽  
Insulin Degrading Enzyme ◽  
Muscle Enzyme ◽  
Porcine Muscle ◽  
Tryptic Fragment ◽  
Human Red Blood Cells ◽  
Subunit Size

The specificity of insulin proteinase (EC 3.4.99.45) has been difficult to categorize using only its natural substrates. By exploiting the fact that two substrates competing for the same enzyme inhibit one another, we have found some new substrates of the insulin proteinase from porcine muscle. Two of these substrates, a tryptic fragment of BSA and a fragment of cytochrome c, have been shown to be cleaved at a single site. The albumin fragment, as well as another fragment of cytochrome c., have susceptibilities (Vmax/Km) comparable with that of insulin. In a second aspect of the study, the porcine-muscle enzyme was shown to be related to other members of its superfamily in that it was immunoprecipitated by a monoclonal antibody raised against the insulin-degrading enzyme from human red blood cells and has the same cleavage sites on insulin as has the rat skeletal-muscle insulin proteinase. We note, however, a possible discrepancy between our results and those of another group regarding the subunit size (110 kDa) of the immunoprecipitated material.

scholarly journals Purification of native p94, a muscle-specific calpain, and characterization of its autolysis

1998 ◽  
Vol 335 (3) ◽  
pp. 589-596 ◽  
Author(s):  
Kayoko KINBARA ◽  
Shoichi ISHIURA ◽  
Shigeo TOMIOKA ◽  
Hiroyuki SORIMACHI ◽  
Seon-Yong JEONG ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cysteine Residue ◽  
Affinity Column ◽  
Cleavage Sites ◽  
Muscle Enzyme ◽  
Active Site Cysteine ◽  
Its Gene ◽  
And Function ◽  
Active Site Cysteine Residue

p94, a skeletal muscle-specific calpain, has attracted much attention because its gene is responsible for limb-girdle muscular dystrophy type 2A. p94, however, has not been characterized at the protein and enzyme levels, owing to its very rapid autolysis. In the present study, a purification procedure for p94 was first established by using a recombinant inactive p94 expressed in COS cells in which the active site cysteine residue was changed to serine [p94(C129S)]. The isolation of native p94 from rabbit skeletal muscle by the established method with conventional procedures was extremely difficult because p94 became highly unstable in a crude extract on the addition of NaCl for separation. Purification of native p94 was possible with an antibody-affinity column but only as an inactive enzyme; p94(C129S) was purified as a homodimer. Characterization of p94, especially autolysis, was performed with partly purified native p94 and p94(C129S). The autolysis of p94, which consisted at least partly of an intermolecular reaction, proceeded in three consecutive steps; 60 and 58 kDa fragments were produced as intermediates before a stable 55 kDa fragment appeared. Autolysis of p94 was regarded as a degradative step rather than for the activation of the enzyme. All the autolysis cleavage sites were located in the p94-specific insertion sequence 1 region, which explains why p94 is unstable compared with the other calpains. The autolysis sites in p94 clearly showed a different specificity relative to the autolytic and proteolytic cleavage sites of the ubiquitous µ- and m-calpains, in its preference for residues at the P3 to P1´ sites, indicating a distinct substrate specificity and function for the muscle enzyme.

scholarly journals Characterization of yeast iso-1-cytochrome c mRNA.

1980 ◽  
Vol 255 (18) ◽  
pp. 8623-8628
Author(s):  
J.M. Boss ◽  
M.D. Darrow ◽  
R.S. Zitomer
Keyword(s):  
Cytochrome C

Effect of training on insulin binding to rat skeletal muscle sarcolemmal vesicles

1986 ◽  
Vol 250 (5) ◽  
pp. E570-E575
Author(s):  
G. K. Grimditch ◽  
R. J. Barnard ◽  
S. A. Kaplan ◽  
E. Sternlicht
Keyword(s):  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Exercise Training ◽  
Insulin Binding ◽  
Whole Body ◽  
Rat Skeletal Muscle ◽  
Binding Studies ◽  
Muscle Enzyme ◽  
Intravenous Glucose ◽  
High Affinity

We examined the hypothesis that the exercise training-induced increase in skeletal muscle insulin sensitivity is mediated by adaptations in insulin binding to sarcolemmal (SL) insulin receptors. Insulin binding studies were performed on rat skeletal muscle SL isolated from control and trained rats. No significant differences were noted between groups in body weight or fat. An intravenous glucose tolerance test showed an increase in whole-body insulin sensitivity with training, and specific D-glucose transport studies on isolated SL vesicles indicated that this was due in part to adaptations in skeletal muscle. Enzyme marker analyses revealed no differences in yield, purity, or contamination of SL membranes between the two groups. Scatchard analyses indicated no significant differences in the number of insulin binding sites per milligram SL protein on the high-affinity (15.0 +/- 4.1 vs. 18.1 +/- 6.4 X 10(9)) or on the low-affinity portions (925 +/- 80 vs. 884 +/- 106 X 10(9)) of the curves. The association constants of the high-affinity (0.764 +/- 0.154 vs. 0.685 +/- 0.264 X 10(9) M-1) and of the low affinity sites (0.0096 +/- 0.0012 vs. 0.0102 +/- 0.0012 X 10(9) M-1) also were similar. These results do not support the hypothesis that the increased sensitivity to insulin after exercise training is due to changes in SL insulin receptor binding.

scholarly journals Crystallization and characterization of Chromatium vinosum cytochrome c'.

1990 ◽  
Vol 265 (10) ◽  
pp. 5364-5365
Author(s):  
D E McRee ◽  
S M Redford ◽  
T E Meyer ◽  
M A Cusanovich
Keyword(s):  
Cytochrome C ◽  
Chromatium Vinosum
Sign in / Sign up

Export Citation Format

Share Document