Degradation of ubiquitin in beef during storage

M. Sekikawa, K. Seno and M. Mikami

Abstract

Sarcoplasmic proteins were prepared from the quadriceps femoris muscle immediately after slaughter (2.5 h) and from stored muscle samples at 10 days postmortem for SDS-PAGE analysis and Western blotting. Characterization with ubiquitin antiserum (Sigma, USA) showed clear and strong recognition of ubiquitin (8.6 kDa) and another minor band (17 kDa) in purified ubiquitin (Sigma, USA). Among the sarcoplasmic proteins prepared, this antiserum also reacted with the bands corresponding to purified ubiquitin (8.6 kDa and 17 kDa) and a small amount of some other higher-molecular-mass proteins which were considered to be ubiquitin-protein conjugates. However, at 10 days postmortem, both ubiquitin and the ubiquitin-protein conjugates had almost disappeared, suggesting their degradation by proteinases.

Introduction

Although it is well known that low-molecular-mass peptides and free amino acids accumulate in the sarcoplasmic fraction of various meats during conditioning (Field et al., 1971, Nishimura et al.,1988, Sekikawa et al., 1996), their structures and sources have not been definitively identified. However, it is considered that this peptide and amino acid accumulation is due to the action of proteases (Dransfield, 1994, Etherington et al., 1990, Koohmarie, 1994, Koohmarie et al., 1986, Mikami et al., 1994).This intracellular protein degradation by different proteolytic mechanisms can be classified into both lysosomal and cytoplasmic (non-lysosomal) pathways in living cells (Ciechanover et al., 1990). In the cytoplasmic pathway, there are ATP-dependent and independent mechanisms. Recent studies have suggested that the ubiquitin system, consisting of ATP, proteasomes and ubiquitin, plays an important role in the degradation of muscle proteins under various catabolic conditions (Taillandier et al., 1996). Ubiquitin is conjugated with proteins targeted for degradation. The ubiquitin molecule is a single polypeptide chain containing 76 amino acid residues, and its amino acid sequence has been highly conserved during evolution (Ciechanover et al., 1990). Another common property of this peptide is its extremely high stability over a wide range of pH and temperature, as well as against protease digestion. Although the ubiquitin system of cellular protein degradation has been investigated in various fields, such as clinical medicine and cellular biology (Fang, C. et al., 1995), this peptide seems to have received little attention in the field of meat science. The purposes of this study were to demonstrate electrophoretically that ubiquitin is present in postmortem skeletal muscle and to study the degradation of this peptide during conditioning.

Materials and Methods

Samples of quadriceps femoris muscle were obtained from two Japanese Brown steers (aged 24 mo) within 2.5 h after slaughter, and the excess fat and fascia were removed. Sarcoplasmic proteins were prepared from the muscle samples immediately after slaughter and at 10 days postmortem after storage at 4±1℃. These samples were homogenized with twice their weight of distilled water, boiled for 5 min, and then allowed to cool to room temperature. Precipitates were removed by centrifugation (0℃, 6,000×g, 30 min) and the supernatant was freeze-dried. The freeze-dried sample obtained was dissolved in distilled water (1:25 w/v) and dialyzed against distilled water at 4±1℃ for 24 h. The dialysate obtained was freeze-dried again, and the final lyophilized sample was considered to be the sarcoplasmic fraction in the present study. The prepared sarcoplasmic fractions were analyzed in a 15% Tris-glycine slab gel with a 6% stacking gel (Laemmli, 1970). The gel was run at 20 mA for 2 h and then stained with CBB G-250 in 30% methanol and 10% acetic acid. Proteins were transferred from the slab gel to a nitrocellulose membrane (ADVANTEC, Japan) by a buffer transfer method (Negishi et al., 1996). After transfer, the membrane was incubated in a solution containing 0.5 mol monoethanolamine and NaCl, pH 8.3, for 10 h at 37 ℃ and then washed three times in phosphate-buffered saline (PBS, pH 7.4) for 5 min at room temperature. The transferred membrane was incubated with ubiquitin antiserum (Sigma, USA) for 1 h at 37 ℃, washed three times in PBS, and then incubated with peroxidase-labeled goat anti-rabbit secondary antibody (BIO-RAD, USA) for 1 h at 37 ℃, followed by two washes in PBS. The peroxidase was detected using 0.2% (w/v) 4-chloro-1-naphthol, 60% ethanol, and 0.01% H2O2.

Results and Discussion

The average pH of the sample was 6.58 at 2.5 h postmortem and 5.74 at 10 days postmortem, indicating that the muscle samples used were biochemically normal. It is well known that low-molecular-mass peptides and free amino acids accumulate in the sarcoplasmic fraction of various meats during conditioning (Field et al., 1971, Nishimura et al.,1988, Sekikawa et al., 1996). This tendency was observed in the SDS-PAGE profiles of the sarcoplasmic proteins, especially under the 17-kDa band (A, lanes 1 and 2). This degradation was considered to be due to certain proteases; calpain, lysosomal cathepsins, or multicatalytic proteinase complex (Dransfield, 1994, Etherington et al., 1990, Koohmarie, 1994, Koohmarie et al., 1986, Mikami et al., 1994).  Recent studies have suggested that the ubiquitin system, consisting of ATP, proteasomes and ubiquitin, plays an important role in the degradation of muscle proteins under various catabolic conditions (Taillandier et al., 1996). This protein is involved in various cellular functions; regulation of intracellular protein degradation, cell cycle regulation and the stress response (Fang, C. et al., 1995, Taillandier et al., 1996). The ubiquitin is covalently ligated to the target protein through an isopeptide linkage between the ubiquitin C-terminal Gly residue and theε-amino group of Lys residues of the targeted protein. The ubiquitin or conjugated protein may also be ligated to additional ubiquitin molecules to form branched poly-ubiquitin. Protein ligated to multiple units of ubiquitin is degraded by the 26S proteasome.

Taillandier et al. (1996) reported that nine days of hindlimb suspension resulted in atrophy (55%) and loss of protein (53%) in rat soleus muscle due to markedly increased in protein breakdown. They also suggested that the lysosomal and Ca2+-activated proteinases contribute to the increased muscle proteolysis seen in atrophy and that increased ATP-ubiquitin-dependent proteolysis may be the critical system responsible for the degradation of myofibrillar proteins in unweighted rats. Although the ubiquitin system of cellular protein degradation has been investigated in various fields, such as clinical medicine and cellular biology (Fang, C. et al., 1995), the state of this peptide in muscle cells postmortem seems to have received little attention.

Characterization of the ubiquitin antiserum showed that it clearly and strongly recognized the ubiquitin band (8.6 kDa) and another minor band (17 kDa) in the purified ubiquitin sample (Sigma, USA; Fig. 1, B, lane 3).

This antiserum also reacted with bands corresponding to purified ubiquitin (8.6 kDa and 17 kDa) and a small amount of some other higher-molecular-mass proteins (35 kDa), which were considered to be the ubiquitin-protein conjugate (B, lane 1). Moreover, when the 8-kDa band purified by preparative SDS-PAGE (Prep cell, BioRAD) was subjected to amino acid sequence analysis by Edman degradation (model 470, ABI), the sequence of the N-terminal five residues (MQIFV) was the same as that of ubiquitin (Schlesinger et al., 1975). These results suggest that the 8-kDa band in the sarcoplasmic proteins includes ubiquitin as a major component. However, at 10 days postmortem, both ubiquitin and the ubiquitin-protein conjugates had almost disappeared, suggesting their degradation by proteases. The results of Western blotting suggested that both the 8-kDa ubiquitin and ubiquitin-protein conjugates existed and/or were released in the sarcoplasmic fraction of muscle cells immediately after arrest of the circulatory system, and were then degraded into peptides or free amino acids by proteinases during conditioning.

It is generally accepted that accumulation of low-molecular-mass peptides in the sarcoplasmic fraction during conditioning is evident in SDS-PAGE profiles using CBB staining. However, in the sarcoplasmic fraction there might be a decrease in low-molecular-mass components during conditioning, such as the 8-kDa ubiquitin band shown in the present study. It was considered that protein degradation with the ubiquitin system, comprising ubiquitin, ATP and proteasomes, which act in living muscle cells, is also one of the primary factors affecting meat conditioning in the immediate postmortem period, since this system requires ATP. It is also apparent that the mechanism of action of ubiquitin is important not only in meat science, but also in general biochemical studies, such as those investigating ischemia of muscle cells. Therefore, further experiments must be designed to demonstrate the contribution of the ubiquitin system during meat conditioning.

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