Biomedical subjects

G H Geesink

Publications and source records attributed to G H Geesink.

14 recordsLinked to original sources

PubMed2006

Micro-calpain is essential for postmortem proteolysis of muscle proteins.

The objective of this investigation was to test the hypothesis that -calpain is largely responsible for postmortem proteolysis of muscle proteins. To accomplish this objective, we compared proteolysis of known muscle proteins in muscles of wild type and micro-calpain knockout mice during postmortem storage. Knockout mice (n = 6) were killed along with control mice (n = 6). Hind limbs were removed and stored at 4 degrees C. Muscles were dissected at 0, 1, and 3d postmortem and subsequently analyzed for degradation of nebulin, dystrophin, metavinculin, vinculin, desmin, and troponin T. In a separate experiment, hind limb muscles from knockout (n = 4) and control mice (n = 4) were analyzed at 0, 1, and 3 d postmortem using casein zymography to confirm that mu-calpain activity was knocked out in muscle and to determine whether or not m-calpain is activated in murine postmortem muscle. Cumulatively, the results of the first experiment indicated that postmortem proteolysis was largely inhibited in micro-calpain knockout mice. The results of the second experiment established the absence of micro-calpain in the muscle tissue of knockout mice and confirmed the results of an earlier study that m-calpain is active in postmortem murine muscle. The results of the current study show that even in a species in which m-calpain is activated to some extent postmortem, micro-calpain is largely responsible for postmortem proteolysis. This observation excludes a major role for any of the other members of the calpain family or any other proteolytic system in postmortem proteolysis of muscle proteins. Therefore, understanding the regulation of micro-calpain in postmortem muscle should be the focus of further research on postmortem proteolysis and tenderization of meat.

Animals↗

PubMed2005

Effect of processing on turkey meat quality and proteolysis.

Modern processing techniques for turkey involve rapid chilling to slow microbial growth and early deboning of the economically important breast meat. This paper shows that these 2 processes lead to significantly tougher meat with higher cooking losses. The toughening appears to be due to less extensive proteolysis and shortening of the sarcomeres. Calpains I and II and their inhibitor, calpastatin, were quantified in turkey breast. Calpain II was the more common isoform but showed no evidence of activation during aging. In contrast, calpain I and calpastatin activities declined rapidly and were no longer detected 24 h postslaughter. There was no evidence of an association between calpain activity and processing conditions.

Animals↗

PubMed2005

Calpain 3/p94 is not involved in postmortem proteolysis.

Studies on the correlation between expression and/or autolysis of calpain and postmortem proteolysis in muscle have provided conflicting evidence regarding the possible role of calpain 3 in postmortem tenderization of meat. Thus, the objective of this research was to test the effect of postmortem storage on proteolysis and structural changes in muscle from normal and calpain 3 knockout mice. Knockout mice (n = 6) were sacrificed along with control mice (n = 6). Hind limbs were removed and stored at 4 degrees C; muscles were dissected at 0, 1, and 3 d postmortem and subsequently analyzed individually for degradation of desmin. Pooled samples for each storage time and mouse type were analyzed for degradation of nebulin, dystrophin, vinculin, and troponin-T. In a separate experiment, hind-limb muscles from knockout (n = 4) and control mice (n = 4) were analyzed for structural changes at 0 and 7 d postmortem using light microscopy. As an index of structural changes, fiber detachment, cracked or broken fibers, and the appearance of space between sarcomeres were quantified. Cumulatively, the results of the first experiment indicated that postmortem proteolysis of muscle occurred similarly in control and in calpain 3 knockout mice. Desmin degradation did not differ (P > 0.99), and there were no indications that degradation of nebulin, dystrophin, vinculin, and troponin-T were affected by the absence of calpain 3 in postmortem muscle. Structural changes were affected by time postmortem (P < 0.05), but not by the absence of calpain 3 from the muscles. In conclusion, these results indicate that calpain 3 plays a minor role, if any, in postmortem proteolysis in muscle.

Animals↗

PubMed2001

The calpain system in three muscles of normal and callipyge sheep.

Activities of mu- and m-calpain and of calpastatin were measured at four different times during postmortem storage (0, 1, 3, and 10 d) in three muscles from either callipyge or noncallipyge (normal) sheep. The weights of two muscles, the biceps femoris and the longissimus, are greater in the callipyge phenotype, whereas the weight of the infraspinatus is not affected. The activity of m-calpain was greater (P < 0.05) in the biceps femoris and longissimus from callipyge than in those from normal sheep, but it was the same in the infraspinatus in the two phenotypes. The extractable activity of m-calpain did not change (biceps femoris and infraspinatus) or decreased slightly (longissimus) during postmortem storage. Extractable activity of mu-calpain decreased to zero or nearly zero after 10 d postmortem in all muscles from both groups of sheep. The rate of decrease in mu-calpain activity was the same in muscles from the callipyge and normal sheep. At all time points during postmortem storage, calpastatin activity was greater (P < 0.05) in the biceps femoris and longissimus from the callipyge than from the normal sheep, but it was the same in the infraspinatus from callipyge and normal sheep. Calpastatin activity decreased (P < 0.05) in all three muscles from both phenotypes during postmortem storage; the rate of this decrease in the callipyge biceps femoris and longissimus and in the infraspinatus from both the callipyge and normal sheep was slow, especially after the first 24 h postmortem, whereas calpastatin activity in the biceps femoris and longissimus from the normal sheep decreased rapidly. During postmortem storage, the 125-kDa calpastatin polypeptide was degraded, but the 80-kDa subunit of mu-calpain was cleaved only to 76- and 78-kDa polypeptides even though extractable mu-calpain activity declined nearly to zero. Approximately 50 to 60% of total mu-calpain became associated with the nonextractable pellet after 1 d postmortem. The myofibril fragmentation index for the biceps femoris and longissimus from normal sheep increased significantly during postmortem storage. The fragmentation index for the infraspinatus from the callipyge and normal sheep increased to an intermediate extent, whereas the index for the biceps femoris and longissimus from the callipyge did not change during 10-d postmortem storage. The results suggest that postmortem tenderization is related to the rate of calpastatin degradation in postmortem muscle and that calpastatin inhibition of the calpains in postmortem muscle is modulated in some as yet unknown manner.

Animals↗

PubMed2001

Properties of myofibril-bound calpain activity in longissimus muscle of callipyge and normal sheep.

Properties of the calpain bound to myofibrils in longissimus muscle from callipyge or noncallipyge sheep were examined after 0, 1, 3, and 10 d of postmortem storage at 4 degrees C. Western analysis has shown that most of this calpain is mu-calpain, although the sensitivity of the antibodies used in the earlier studies could not eliminate the possibility that up to 10% of the calpain was m-calpain. The calpain is bound tightly, and very little is removed by washing with the detergent Triton X-100; hence, it is not bound to phospholipids in the myofibril. Over 25% of total mu-calpain was bound to myofibrils from at-death muscle, and this increased to approximately 40% after 1 d postmortem. The amount of myofibril-bound mu-calpain increased only slightly between 1 and 10 d of postmortem storage. The percentage of autolyzed mu-calpain increases with time postmortem until after 10 d postmortem, when all myofibril-bound mu-calpain is autolyzed. The specific activity of the myofibril-bound calpain is very low and is only 6 to 13% as high as the specific activity of extractable mu-calpain from the same muscle. It is unclear whether this low specific activity is the result of unavailability of the active site of the myofibril-bound calpain to exogenous substrate. The myofibril-bound calpain degrades desmin, nebulin, titin, and troponin T in the myofibrils, and also releases undegraded alpha-actinin and undergoes additional autolysis when incubated with Ca2+; all these activities occurred slowly considering the amount of myofibril-bound calpain. Activity of the myofibril-bound calpain was partly (58 to 67%) inhibited by the calpain inhibitors, E-64 and iodoacetate; was more effectively inhibited by a broader-based protease inhibitor, leupeptin (84 to 89%); and was poorly inhibited (43 to 45%) by calpastatin. Release of undegraded alpha-actinin and autolysis are properties specific to the calpains, and it is unclear whether some of the myofibril-bound proteolytic activity originates from proteases other than the calpains or whether the active site of myofibril-bound calpain is shielded from the inhibitors. Activities and properties of the myofibril-bound calpain were identical in longissimus muscle from callipyge and normal sheep, although previous studies had indicated that the "normal" longissimus was much more tender than the callipyge longissimus. Hence, it seems unlikely that the myofibril-bound calpain has a significant role in postmortem tenderization of ovine longissimus.

Animals↗

PubMed2000

Rigor temperature and meat quality characteristics of lamb longissimus muscle.

The present experiment was conducted to determine the effect of muscle temperature during the prerigor and early postrigor period on meat tenderness, postmortem proteolysis, calpain system activity, water-holding capacity, and color. Lamb longissimus muscle (n = 14) from the right and left carcass sides was excised immediately after dressing, divided into an anterior and posterior sample, vacuum-packaged, and stored overnight at 5 to 35 degrees C. Further storage, up to 14 d postmortem, was at 2 degrees C. Tenderness at 1 d postmortem, tenderization during further storage, and postmortem proteolysis were negatively affected by overnight incubation above 25 degrees C. This effect could be explained by an effect of temperature on muscle contraction and activity of the calpain system. Muscle contraction was at a minimum after incubation at 15 degrees C. Water-holding capacity was negatively affected by incubation above 25 degrees C. Color scores improved with increasing incubation temperature at 1 d postmortem. However, after 14 d of postmortem storage, no differences in color scores were observed. Based on the present results and results of other groups, a temperature around 15 degrees C at the onset of rigor seems optimal to maximize tenderness without having detrimental effects on water-holding capacity or color.

Animals↗

PubMed2000

Ionic strength-induced inactivation of mu-calpain in postmortem muscle.

The present study was conducted to study the stability of autolyzed mu-calpain activity and determine whether measurement of mu-calpain activity after anion exchange chromatography accurately reflects its activity in postmortem muscle. Ionic strength and pH affected the stability of partially autolyzed mu-calpain. Complete loss of activity was observed as a result of binding of autolyzed mu-calpain to DEAE-Sephacel when the large subunit of mu-calpain was autolyzed from 80 to 76 kDa. Therefore, determination of mu-calpain by standard anion exchange chromatography may underestimate mu-calpain activity in postmortem muscle. The activity of autolyzed mu-calpain was stabilized by inclusion of glycerol in the buffers, and this permitted us to investigate whether the apparent loss of mu-calpain activity in postmortem muscle is an artifact of the methodology. Despite the inclusion of glycerol in the buffers, a decrease in mu-calpain activity was observed during postmortem storage of muscle, even though the autolyzed enzyme was readily detectable by Western blotting in muscle extracts and column eluates. This result indicates that instability of autolyzed mu-calpain is a major cause for the decline in mu-calpain activity in postmortem muscle.

Animals↗

PubMed1999

Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions.

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.

Animals↗

PubMed1999

Technical note: a rapid method for quantification of calpain and calpastatin activities in muscle.

Stepwise and continuous gradient ion-exchange chromatography were compared for yield of calpains and calpastatin from ovine muscle in a study designed to quantify their activities for comparative purposes. In Exp. 1, a continuous (25 to 400 mM NaCl) gradient and a two-step gradient method (200 mM NaCl to coelute mu-calpain and calpastatin together and then 400 mM NaCl to elute mu-calpain) were compared. For the two-step method, mu-calpain activities were determined by subtracting calpastatin activities before and after heat inactivation of mu-calpain. Both the two-step and the continuous gradient method yielded similar results over a broad range of activities. The stepwise gradient method does not require the use of fraction collectors and pumps, and it can be completed in a fraction of the time required for the continuous gradient method. In Exp. 2, the two-step method was compared with a three-step method (100 mM NaCl to elute calpastatin, then 200 mM NaCl to elute mu-calpain, and then 400 mM NaCl to elute m-calpain). Unlike the continuous gradient method, calpastatin and mu-calpain could not be completely separated using the three-step chromatography method. Thus, the three-step gradient method should not be used to quantify the components of the calpain proteolytic system. The present results indicate that the two-step gradient method is a fast and inexpensive method to determine calpain and calpastatin activities in studies designed to quantify the components of the calpain proteolytic system in skeletal muscle.

Animals↗

PubMed1999

Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb biceps femoris during extended postmortem storage.

The present experiment was conducted to determine whether calpastatin inhibits only the rate, or both the rate and extent, of calpain-induced postmortem proteolysis. Biceps femoris from normal (n = 6) and callipyge (n = 6) lamb was stored for 56 d at 4 degrees C. Calpastatin activity was higher (P < .05) in the callipyge muscle at 0 and 14 d postmortem, but not at 56 d postmortem. The activity of mu-calpain did not differ between normal and callipyge biceps femoris at 0 and 56 d postmortem (P > .05), but was higher at 14 d postmortem in the callipyge muscle (P < 0.05). The activity of m-calpain was higher in the callipyge muscle (P < 0.05). Western blot analyses of titin, nebulin, dystrophin, myosin heavy chain, vinculin, alpha-actinin, desmin, and troponin-T indicated that postmortem proteolysis was less extensive in callipyge than in normal biceps femoris at all postmortem times. The results of this experiment indicate that calpastatin inhibits both the rate and extent of postmortem proteolysis.

Actinin↗

PubMed1998

An improved purification protocol for heart and skeletal muscle calpastatin reveals two isoforms resulting from alternative splicing.

Employment of a new protocol for efficient purification of calpastatin revealed the presence of two forms of calpastatin in porcine heart with apparent molecular weights of 125 and 145 kDa. Purification from bovine muscle resulted in a single species with an apparent molecular weight of 125 kDa. The presence of multiple species of calpastatin in porcine heart does not appear to be an artifact of the purification procedure since Western blotting revealed the presence of two types of calpastatin in ovine and porcine heart, but only one type in bovine heart and bovine, ovine, and porcine muscle. The origin of the two species in porcine heart was examined by RT-PCR and direct sequencing of calpastatin cDNA. This analysis revealed that porcine skeletal muscle exclusively produces transcripts lacking exon 3, while porcine heart produces transcripts that include or lack exon 3, consistent with the presence of two isoforms of the protein. The 125-kDa form of porcine calpastatin therefore appears to be a result of alternative splicing of the calpastatin transcript. The biological significance of the heart specific isoform is not clear; however, its ability to inhibit micro- or m-calpain does not differ considerably. The present purification protocol yielded 4.9 and 1.8 mg calpastatin per kg tissue from porcine heart and bovine skeletal muscle, respectively.

Alternative Splicing↗

PubMed1995

Is Z-disk degradation responsible for postmortem tenderization?

A number of studies have suggested that Z-disk degradation is a major factor contributing to postmortem tenderization. These conclusions seem to have been based largely on experimental findings showing that the calpain system has a major role in postmortem tenderization, and that when incubated with myofibrils or muscle strips, purified calpain removes Z-disks. Approximately 65 to 80% of all postmortem tenderization occurs during the first 3 or 4 d postmortem, however, and there is little or no ultrastructurally detectable Z-disk degradation during this period. Electron microscope studies described in this paper show that, during the first 3 or 4 d of postmortem storage at 4 degrees C, both costameres and N2 lines are degraded. Costameres link myofibrils to the sarcolemma, and N2 lines have been reported to be areas where titin and nebulin filaments, which form a cytoskeletal network linking thick and thin filaments, respectively, to the Z-disk, coalesce. Filamentous structures linking adjacent myofibrils laterally at the level of each Z-disk are also degraded during the first 3 or 4 d of postmortem storage at 4 degrees C, resulting in gaps between myofibrils in postmortem muscle. Degradation of these structures would have important effects on tenderness. The proteins constituting these structures, nebulin and titin (N2 lines); vinculin, desmin, and dystrophin (three of the six to eight proteins constituting costameres); and desmin (filaments linking adjacent myofibrils) are all excellent substrates for the calpains, and nebulin, titin, vinculin, and desmin are largely degraded within 3 d postmortem in semimembranosus muscle. Electron micrographs of myofibrils used in the myofibril fragmentation index assay show that these myofibrils, which have been assumed to be broken at their Z-disks, in fact have intact Z-disks and are broken in their I-bands.

Animals↗

PubMed1993

Effects on meat quality of the use of clenbuterol in veal calves.

The objective of this study was to examine the effects of clenbuterol administration on meat quality traits of veal. Sixteen Holstein-Friesian veal calves (male) were randomly assigned to one of four treatment groups; either control (n = 4) or clenbuterol-treated (1.6 micrograms.kg [corrected] BW-1.d-1, 42 d) with a withdrawal period between clenbuterol treatment and slaughter of 8 d (n = 4), 4 d (n = 4), or 2 d (n = 4). All animals were slaughtered at the same day at a commercial slaughterplant. At 30 min postmortem the carcasses were split and the right carcass side was electrically stimulated. After 24 h of cooling the longissimus, semimembranosus, triceps brachii, and psoas major muscles were excised and vacuum-packaged. After 1, 7, and 13 d of vacuum storage at 2 +/- 2 degrees C the muscles were sampled to determine tenderness, water-holding capacity, and color characteristics. Clenbuterol treatment resulted in a slower rate of pH decline in the unstimulated longissimus muscle but did not affect the ultimate pH. Clenbuterol treatment resulted in toughening of the longissimus, semimembranosus, and triceps brachii muscles after 1 and(or) 7 d of storage (P < .05). It is suggested that this resulted from a decrease in postmortem proteolysis because both the intensity of a 30-kDa peptide and the myofibril fragmentation index were lower in clenbuterol-treated muscles. Clenbuterol treatment resulted in increased lightness (L*-value) of longissimus and semimembranosus muscles (P < .05), coincident with a lower water-holding capacity. In a following experiment, the effect of clenbuterol administration (0 [n = 5] and 1.0 [n = 5] mg/kg of feed for 27 d) on calpain and calpastatin levels at 1 d postmortem in longissimus muscles of Friesian Pie Noire veal calves was investigated. Clenbuterol administration resulted in an increase in calpastatin levels (P < .05) and a trend (P < 0.1) toward a decrease in mu-calpain activity at 1 d postmortem.

Animals↗

PubMed1992

The role of ultimate pH in proteolysis and calpain/calpastatin activity in bovine muscle.

Of a total of three Friesian cows, two of which had been treated with adrenalin before slaughter, Mm longissimus (LO), supraspinatus (SS), triceps brachii (TB) and rectus abdominis (RA) were sampled at different times post mortem (pm). pH, calpain/calpastatin activities and degradation of myofibrillar proteins, as evidenced by SDS-PAGE, were assessed. Contraction characteristics were measured by determining myofibrillar ATPase activities. Adrenalin treatment resulted in a high ultimate pH (6.48 +/- 0.40) and a faster decline pm of calpain I activity. The effect was similar in all four investigated muscles (72.4 +/- 5.4% decline at 24 h pm). The decline in calpain I activity in the control muscles was muscle-dependent and ranged from 22.8-74.3% at 24 h pm. Differences in ultimate pH did not lead to distinct rates of breakdown of proteins with molecular weights lower than that of myosin heavy chain. Calpastatin levels were muscle-dependent and correlated with myofibrillar ATPase activity (r = -0.99). In a second experiment Mm rectus abdominis (RA) and psoas major (PM) of adrenalin-treated (n = 6) and control (n = 6) Friesian-Holstein calves were sampled at 1 and 29 h pm for assessment of calpain activities. At seven days pm the M longissimus (LO) was sampled for tenderness evaluation. pH values were measured at 30 min, 4 h and 29 h pm. Adrenalin treatment resulted in a higher ultimate pH in the three muscles. Higher ultimate pH resulted in lower calpain activities in the RA at 29 h pm (P less than or equal to 0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗