Carlos H. Herrera-Mendez

Revisiting the conversion of muscle into meat and the underlying mechanisms

The conversion of muscle into meat is a complex process in which all mechanisms responsible for the development of meat qualities are very likely interdependent. Colour and flavour are thus both dependent on oxidative mechanisms. Oxidation and proteolysis are probably two processes involved in the development of meat tenderness. This paper reviewed the consequences of programmed cell death or apoptosis on muscle cells structure and biochemistry and on meat qualities as well. We therefore look at different new hypothesis susceptible to highlight the meat science field and provide new supports for a more dynamic meat research. One of them which would have appeared evident for our purpose since a decade, deals with the fact that, after animal bleeding, muscle cells have no other alternative to only enter the programmed cell death procedure or apoptosis. If we introduce an early phase corresponding to apoptosis, taking place before the rigor onset and overlapping it, we will see that the known consequences of that process bring forward possible answers to still unexplained observations. After an overview of the actual state-of-the-art in meat science, we will introduce the programmed cell death and its underlying mechanisms. We then described the strong analogies between the known consequences of apoptosis and the postmortem changes affecting a set of different muscle characteristics.

Muscle endopin 1, a muscle intracellular serpin which strongly inhibits elastase: purification, characterization, cellular localization and tissue distribution

In the present work, an endopin-like elastase inhibitor was purified for the first time from bovine muscle. A three-step chromatography procedure was developed including successively SP-Sepharose, Q-Sepharose and EMD-DEAE 650. This procedure provides about 300 microg of highly pure inhibitor from 500 g of bovine diaphragm muscle. The N-terminal sequence of the muscle elastase inhibitor, together with the sequence of a trypsin-generated peptide, showed 100% similarity with the cDNA deduced sequence of chromaffin cell endopin 1. Hence, the muscle inhibitor was designated muscle endopin 1 (mEndopin 1). mEndopin 1 had a molecular mass of 70 kDa, as assessed by both gel filtration and SDS/PAGE. According to the association rates determined, mEndopin 1 is a potent inhibitor of elastase (kass=2.41x10(7) M(-1).s(-1)) and trypsin (kass=3.92x10(6) M(-1).s(-1)), whereas plasmin (kass=1.78x10(3) M(-1).s(-1)) and chymotrypsin (kass=1.0x10(2) M(-1).s(-1)) were only moderately inhibited. By contrast, no inhibition was detected against several other selected serine proteinases, as well as against cysteine proteinases of the papain family. The cellular location of mEndopin in muscle tissue and its tissue distribution were investigated using a highly specific rabbit antiserum. The results obtained demonstrate an intracellular location and a wide distribution in bovine tissues.

Calpain 1 Binding Capacities of the N1-Line Region of Titin Are Significantly Enhanced by Physiological Concentrations of Calcium†

Calpain 1, an ubiquitous well-known calcium-dependent intracellular protease, was recently shown to bind tightly to the proximal end of the I-band titin segment in a calcium-dependent manner [Raynaud et al. (2005) FEBS J. 272, 2578-2590]. In the present work we identified the titin Ig-domain of concern by this interaction and the role of calcium in this interaction using a recombinant fragment of titin spanning the I2-I6 region and its subfragments. The heterodimeric form of calpain 1 binds to this titin fragment with a very high affinity ( K d = 5.1 +/- 0.2 x 10 (-7) M) at much lower calcium levels than those saturating the high-affinity binding sites of the peptidase ( K d = 25 microM). Investigation of this interaction with I2-I6 subfragments clearly showed that the dimeric form of calpain 1 binds exclusively to the Ig-domain I4 of titin with an affinity similar to that of the whole I2-I6 segment. As for the I2-I6 fragment, this interaction is calcium regulated. Calcium was shown to bind tightly to titin ( K d = 1.9 x 10 (-7) M), causing an oligomerization of the titin segment. At physiological calcium concentration (10 (-6) to 10 (-8) M), the prevailing form of the titin fragment is a trimer, suggesting that calpain 1 binds to this titin structure. From the present findings, it was concluded that calcium binding to titin increased the amount of bound calpain 1 (up to 40% of the total calpain 1) and that this bound calpain 1 might constitute a reservoir for this peptidase. In this context, we proposed a schematic diagram of this series of calcium-dependent events with the inherent unanswered questions. These events are probably under a complex regulation involving undoubtedly different yet unidentified proteins.

Inhibition of human initiator caspase 8 and effector caspase 3 by cross-class inhibitory bovSERPINA3-1 and A3-3

MINT‐7234625: SERPINA3‐1 (uniprotkb:Q9TTE1) and CASP3 (uniprotkb:P42574) bind (MI:0407) by biochemical (MI:0401)

Purification of the skeletal muscle protein Endopin 1B and characterization of the genes encoding Endopin 1A and 1B isoforms

In the present work, a new endopin‐like serpin designed mEndopin 1B was purified from bovine muscle. Biochemical characterizations (amino acid sequencing and Maldi‐Tof mass spectrometry peptide mapping) demonstrated that the purified protein is different from the previously described Endopin 1, renamed mEndopin 1A. The genes and cDNA of both endopins were characterized. The cDNA sequence of mEndopin 1B encodes a predicted protein of 411 amino‐acids with a molecular mass of 43 808 Da. The mEndopin 1B gene comprised four coding exons and an additional 5′ untranslated exon. The reactive site sequence of mEndopin 1B is somewhat different from that of mEndopin 1A. Nevertheless, both serpins have a similar peptidase inhibitory pattern against examined proteases (elastase, trypsin, plasmin and chymotrypsin). The high expression of both mEndopin 1A and 1B in bovine serum and tissues and their high efficiency to inhibit elastase (k ass ∼ 106 − 107 M−1 s−1) suggested that these serpins might play a major role in inflammatory processes.

Serine Protease Inhibitors as Good Predictors of Meat Tenderness: Which Are They and What Are Their Functions?

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990s, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50–70 kDa), F2 (Mr:40–60 kDa) and F3 (Mr:10–15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.

Cells Shrinkage and Phosphatidylserine Externalization in Post Mortem Muscle by Fluorescence Microscopy

Postmortem (PM) meat tenderization is a complex biochemical process that involves multiple endogenous proteolytic enzymes and that is not completely understood. Cell death by apoptosis is recently proposed as a novel mechanism in this process. The signification of the caspases implication in muscle proteins degradation is that the cells are dying through apoptosis, a major cell death programme. Our purpose was therefore to verify this statement using different well known hallmarks of the apoptotic process including cell shrinkage, phosphatidylserine (PS) externalization. Cells start to shrink few minutes after animal death and reach a maximum shrinkage about 24 h postmortem while they showed the typical rounding appearance of apoptotic cells. Externalization of PS is detectable within 1 h postmortem and increased gradually with time. The present work is therefore the first to provide direct evidence supporting the onset of apoptosis in post-mortem muscle. The present findings should lead us to reconsider as a whole, the mechanisms contributing of the development of meat qualities (tenderness, flavor, juiciness …) by a detailed analysis of the apoptosis associated biochemical and physiological modifications taking place within the cells.