Akihiro Okitani

Mode of degradation of myofibrillar proteins by an endogenous protease, cathepsin L

The mode of degradation of myofibrils and their constituent proteins by cathepsin L (EC 3.4.22.15) of rabbit skeletal muscle was studied. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that cathepsin L degraded myosin heavy chain, alpha-actinin, actin, troponin T and troponin I assembled in myofibrils and produced mainly fragments of 160 000 and 30 000 daltons in the acidic pH region. This degradation was most intense around pH 4. Degradation of myosin in the isolated state by cathepsin L resulted in the disappearance of the heavy chain and the decrease of light chains 1, 2 and 3, producing fragments of 160 000, 92 000, 83 000 and 60 000 daltons. The degradation of the heavy chain was most severe at pH 4.2. Cathepsin L degraded actin into fragments of 40 000, 37 000 and 30 000 daltons. This action was most intense at pH 4.7. Tropomyosin was not degraded. Troponin T and troponin I were degraded into fragments of 30 000 and 13 000 daltons at pH 3.7--6.7, which were degraded further into smaller fragments. Troponin C was not degraded. alpha-Actinin was degraded into several fragments, the major one of which showed an Mr of 80 000. This degradation was most intense at pH 3.0--3.5.

Purification of cathepsin D from rabbit skeletal muscle and its action towards myofibrils

Cathepsin D (EC 3.4.23.4) was purified from rabbit skeletal muscle using acetone-dried muscle powder as starting material. After the acetone-dried powder was extracted with 0.2 mM ATP, the extract was fractionated with acetone an subjected to DEAE-Sephadex A-50 and Sephadex G-100 column chromatography. Rechromatography on a Sephadex G-100 column resulted in a purified preparation. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed one major band of 42,000 daltons and some bands of contaminants. Since gel filtration also indicated a value of 42,000 daltons for the enzyme, it was concluded that muscle cathepsin D has no subunit structure. The enzyme acted optimally towards myofibrils around pH3, resulting in the degradation of the myosin heavy chain and production of a 30,000-dalton component.

Mode of degradation of myofibrillar proteins by rabbit muscle cathepsin D

The mode of degradation of myofibrillar proteins by the action of highly purified rabbit muscle cathepsin D (EC 3.4.23.5) was studied using SDS-polyacrylamide gel electrophoresis. Cathepsin D optimally degraded myosin heavy chain, alpha-actinin, tropomyosin, troponin T and troponin I at around pH 3. It did not degrade actin or troponin C. Degradation of myosin heavy chain produced four major fragments of 155000, 130000, 110000 and 90000 daltons. Troponin T was hydrolyzed to 33000-, and 20000- and 11000-dalton fragments. Troponin I was degraded into fragments of 13000 and 11000 daltons. Degradation of alpha-actinin and tropomyosin was not as rapid as that of myosin and troponins T and I. Tropomyosin gave a fragment of 30000 daltons, but alpha-actinin showed no distinct band of this fragment on gels.

Morphological changes in myofibrils and glycerinated muscle fibers on treatment with cathepsins D and L.

Morphological changes in myofibrils and glycerinated muscle fibers on treatment with cathepsins D and L obtained from rabbit skeletal muscle were studied by phase contrast and electron microscopy. Cathepsin D degraded the Z-line and filaments adjacent to the H-zone. Cathepsin L caused the fragmentation of myofibrils, and degraded the Z-line and M-line. Both cathepsins induced disturbance of the lateral arrangement of myofibrils.

Cysteine proteinase inhibitors from rabbit skeletal muscle

1. Two cysteine proteinase inhibitors, I-T (Mr = 29,000) and I-S (Mr = 10,700), were isolated from rabbit skeletal muscle by means of succesive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75. 2. I-T is a formed trimer of a monomeric inhibitor, I-M (Mr = 10,500), through disulfide bonds. 3. I-S is almost completely stable between pH 3 and 8, while I-M is unstable in the same pH range. 4. I-M acts most effectively towards cathepsins H and L, showing moderate activity towards cathepsin B and only weak activity towards papain. I-S acts most effectively towards cathepsin L, followed by, in decreasing order, cathepsin H, cathepsin B and papain.

Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle

Two cysteine proteinase inhibitors, CPI-L and CPI-H, were purified from rabbit skeletal muscle by means of successive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. The molecular mass of CPI-L was 13 kDa on gel permeation chromatography and SDS-PAGE under reducing conditions and was 15 kDa on SDS-PAGE under non-reducing conditions. The molecular mass of CPI-H was 23 kDa on gel permeation chromatography and it was converted to 13 kDa by SH-reducing agent. Although CPI-H showed single protein band with 13 kDa on SDS-PAGE under reducing conditions, it showed four protein bands with 21, 20, 15 and 13 kDa on SDS-PAGE under non-reducing conditions. Therefore, CPI-H was suggested to have a complicated subunit structure for which S-S bonds and some non-covalent bonds would be responsible. CPI-L and CPI-H were stable in the range of pH 3.0-9.5 and up to 80 degrees C. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (Ki) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B.

Studies on Myofibrils from the Stored Muscle:Part I. Post-mortem Changes in Adenosine Triphosphatase Activity of Myofibrils from Rabbit Muscle

Adenosine triphosphatase (ATPase) activity of myofibrils isolated from fresh muscle and the muscle stored at 4°C have been measured.An increase in Mg-activated ATPase activity of myofibrils was caused by lengthened homogenization.With the progress of aging of muscle, Mg-activated ATPase activity of myofibrils increased remarkably.When myofibrils from pre-rigor and rigor muscle in 0.16 m KCl were treated with 0.6 m KCl-18 mm Tris-maleate solution (pH 7.0), Mg-activated ATPase activity of myofibrils at low ionic strength increased markedly. However, the Mg-activated ATPase activity of the myofibril isolated from the muscle stored at 4°C for 8 days (8-myofibril) increased slightly after the similar treatment.The dependence of myofibrillar ATPase activity on KCl concentration became greater with the progress of aging of muscle.These results may show that, as long as ATPase activity and the dependence of ATPase activity on KCl concentration are concerned, 8-myofibril is the most similar to the isolated actomyo...

Purification and some properties of cathepsin H from rabbit skeletal muscle.

Rabbit muscle cathepsin H classified as an aminoendopeptidase was purified and its properties were investigated to clarify its contribution to the proteolysis of postmortem muscle. The purification was performed by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocelluose, DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme gave a single protein band on SDS-PAGE. Its molecular mass was found to be 28 kDa by gel permeation and 30 kDa by SDS-PAGE. The optimum pHs for alpha-N-benzoyl-DL-arginine-beta-naphthylamide (BANA)- and L-leucine-beta-naphthylamide (Leu-NA)-hydrolyzing activities were 6.6 and 7.0, respectively. This enzyme was almost stable in the range of pH 4-5 and up to 50 degrees C at pH 5.0. The Km values of BANA- and Leu-NA-hydrolyzing activities were 0.367 and 0.203 mM, respectively. The enzyme was inhibited by monoiodoacetic acid, antipain, leupeptin, TLCK and TPCK, but was not affected by pepstatin, bestatin, puromycin, PMSF or trypsin inhibitor. This enzyme strongly acted on Arg-, Lys-, Met-, Ala-, Ser- and Leu-NAs, weakly acted on Val- and Glu-NAs, and hardly acted on Pro- and Gly-NAs. The amount of cathepsin H in muscle was estimated to be less than one-fourth of the sum of the amount of aminopeptidases C and H by the Leu-NA-hydrolyzing activity on the chromatography. This enzyme degraded myosin heavy chain, actin, tropomyosin and troponin I clearly at pH 4.0, while it slightly degraded troponin I at pH 5.0-5.6. Therefore, the contribution of cathepsin H to the proteolysis of postmortem muscle is presumed to be relatively small.

Purification and properties of aminopeptidase C from porcine skeletal muscle

1. Aminopeptidase C was purified from porcine skeletal muscle. 2. The mol. wt of the enzyme was found to be 103,000 on both Sephadex G-200 column chromatography and SDS-PAGE. 3. The optimum pH for the hydrolysis of L-leucine p-nitroanilide was around 7.0. 4. The activity of this enzyme was strongly inhibited by EDTA, bestatin and puromycin. 5. The enzyme acted on the beta-naphthylamide derivatives of amino acids and oligopeptides.

Elucidation of a basic protein, glyceraldehyde-3-phosphate dehydrogenase, as a contributing factor to raise Mg-ATPase activity of myofibrils during meat conditioning

We investigated the factor which increased the maximum value of the Mg-ATPase activity of myofibrils existing at low KCl concentrations during meat conditioning. On the treatment of myofibrils with the solution extracted with the buffer of pH 7.2 from muscle, the Mg-ATPase activity in the presence of 0-0.15 M KCl increased time-dependently. This change was most remarkable in the range of pH 5.6-7.0. Trypsin treatment of the extract abolished such effect, suggesting that the responsible factors were proteins. The fractionation of the extract with isoelectric focusing demonstrated that the factors were basic proteins (pI 8.3-9.6). The treatment of myofibrils with those basic proteins under various conditions suggested that the time-dependent adhesion of those basic proteins, through a denaturation at around pH 5.5, to myofibrils was assumed to raise the Mg-ATPase activity. Analysis of myofibrils prepared from rabbit muscles stored at 0°C for 12 days postmortem showed the appearance of the 35,000 Da protein, accompanied the increase in the Mg-ATPase activity. Therefore, the adhesion of this protein to myofibrils in situ probably caused the increase in the Mg-ATPase activity. Successive treatment with the basic protein and the crude cathepsin increased the dependency of the Mg-ATPase activity on KCl concentrations and the maximum value of the Mg-ATPase activity. Therefore, the coordinate action of a basic 35,000 Da protein and cathepsins was presumed to induce the changes in the Mg-ATPase activity of myofibrils during meat conditioning. The basic protein was concluded to be glyceraldehyde-3-phosphate dehydrogenase (its subunit molecular mass: 35,000 Da), since the incubation of myofibrils with its commercial preparation raised the Mg-ATPase activity of myofibrils.