Low temperature activation of post mortem glycogenolysis in bovine skeletal muscle fibres.

Synopsis Trimmed strips of stermomandibularis muscles taken from freshlyslaughtered cattle were placed in an isotonic myograph and cooled to 1°C. Spontaneous activity due to neuromuscular irritability was minimized by keeping muscle surfaces moist and anaerobic and was monitored by electromyography. Muscle strips were removed and frozen for histochemical analysis after they had completed their initial phase of cold-induced shortening (several hours). Control strips maintained for an equal time at 24°C rarely depleted the stainable glycogen in any of their muscle fibres so as to become PAS-negative. In chilled muscle strips, however, glycogenolysis was activated in some muscle fibres and they became PAS-negative. In serial sections, most of the PAS-negative fibres exhibited strong ATPase and weak succinate dehydrogenase activity. Fibres with weak ATPase and strong succinate dehydrogenase activity rarely became PAS-negative. These results are in agreement with biochemical reports of a cold-induced (<5°C) activation of glycolysis in skeletal musclepost mortem. Investigations on untrimmed lengths of excised sternoman dibularis muscle indicated that longitudinal muscle damage caused in cutting muscle strips for the myograph and/or their more rapid rate of initial cooling had facilitated the depletion of stainable glycogen.     

The reversible binding of glycolytic enzymes in ovine skeletal muscle in response to tetanic stimulation

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in sheep hind muscles after electrical stimulation. As compared to the control muscles, stimulation led to significant increases in the amount of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase bound to the particulate fraction. The bindng of other glycolytic enzymes was not significantly altered. A servey of different hind limb muscles at variable rates of stimulation revealed that each muscle exhibited its own characteristic response pattern in terms of the level of increased enzyme binding. Generally, an increased stimulation rate led to greater enzyme adsorption. The increase in enzyme binding was rapidly reversible for it was shown that the amount of enzyme bound quickly returned to control values when the muscles were allowed to recover in the live anaesthetised animal following cessation of stimulation. Those muscles which exhibited increased enzyme binding were characterised by a marked loss of glycogen and accumulation of lactate suggesting that accelerated glycolytic flux was a necessary condition for the observation of increased enzyme binding. In support of this, enzyme adsorption was observed to be greatest on stimulation of ischemic muscles, whereas in trained muscles, or muscles with depleted glycogen stores induced by prior adrenalin treatment, the increased enzyme binding response was greatly diminished. It is concluded that the variable binding of key glycolytic enzymes has a role to play in the regulation of glycolytic behaviour in skeletal muscle.     

Changes in enzymes associated with energy metabolism during the early post mortem period in longissimus thoracis bovine muscle analyzed by proteomics

Changes in metabolic protein levels in biopsies during the early post mortem period in the bovine longissimus thoracis muscle were investigated by 2-DE based proteome analyses. Nine NRF (Norwegian Red) dual purpose bulls were included in the study. Twenty-four proteins underwent changes between the two sampling times and were classified into two major groups: metabolic proteins and heat shock proteins. Of the metabolic proteins, 5 enzymes involved in the glycolytic pathway and the tricarboxylic acid (TCA) cycle, increased in intensities during the post mortem period. In addition, the NADP-dependent enzyme 3-hydroxyisobutyrate dehydrogenase, associated with the TCA cycle in muscle, was increased. This documents that an increased aerobic energy metabolism occurs immediately after slaughter, with the aim to replenish the ATP levels in the muscle.     

Microenvironmental factors and the binding of glycolytic enzymes to contractile filaments.

1. In reviewing the microenvironmental factors involved in the binding of the glycolytic enzymes to contractile filaments, consideration has been given to the significance of molecular crowding in maintaining these interactions under cellular conditions, and the influence of hormones, metabolites, pH and enzyme modifications on these phenomena. 2. Overall, these data serve to emphasize the biological reality of these associations, and their micro-organizational adaptations during physiological activities.     

Effect of triiodothyronine on human jejunal glycolytic enzymes.

Antisera against rat liver aspartate aminotransferase (EC 2.6.1.1) isozymes were used to study the activity and immunologic pattern of these isozymes in the livers of the rat, mouse, hamster, gerbil and in Ehrlich ascites cells. A double immunodiffusion precipitin test and immunoelectrophoresis showed that, except for the gerbil, there was a pattern of identity of AAT isozymes in the presence of either the antianionic or the anticationic antisera. Although gerbil AAT isozymes are immunochemically different from those of the other rodents studied, they were inactivated by the respective antiserum in a manner similar to that observed with the other species. This may suggest that antigenic determinants at the catalytic site of each of the liver aspartate aminotransferase isozymes are least likely to change throughout the evolutionary process.     

The assessment of post mortem structural changes in the human epidermis

The structure of epidermis and appearance of keratinocytes is described in intact skin specimens from human corpses stored after death under refrigeration. Two groups of alterations can be identified depending on the epidermal layer. In the spinous layer, the cells are characterized by crescent-shaped nuclei surrounded by a hollow area. The number of such cells increases significantly each day during the first 8 days post mortem (dpm), and their frequencies follow respective regression equations, so as to enable the post mortem time estimation with one day accuracy. In the basal layer, distorted, balloon-shaped cells with pycnotic nuclei appear, which with the lapse of time are forming groups, and eventually the epidermis in those places separates from the dermis. The presence of both described changes seems to indicate whether the skin sample was obtained from the living organism or after the death.     

Analysis and predication of structural motifs in the glycolytic enzymes

Protein crystallography has determined the three-dimensional structures of 10 of the 13 enzymes of the glycolytic pathway. Diagrams and details of these enzyme structures are given in the paper. Most of the enzyme domains are variations and extensions of a many (4--9)-stranded, predominantly or totally parallel, beta-sheet that is shielded from solvent by alpha-helices (i.e. alpha/beta structures). There are strong structural similarities between the domains of some, but not all, of the enzymes. In particular the dinucleotide binding fold of lactate dehydrogenase and the beta-barrel of triose phosphate isomerase are found in other domains. General rules governing the topology and packing of alpha-helices against a beta-sheet provide a basis for the combinatorial prediction of the tertiary fold of glycolytic domains from their amino acid sequence and observed secondary structure. The predication algorithm demonstrates that there are severe restrictions on the number of possible structures. However, these restrictions do not fully explain some of the remarkable structural similarities between different enzymes that probably result from evolution from a common ancestor.     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.     

The binding of glycolytic enzymes to the cytoskeleton--influence of pH

In a continuing study of the interactions between glycolytic enzymes and cytoskeletal structure, the influence of a variation of the pH of the eluting medium has been investigated. This treatment resulted in an increased degree of binding of most of the glycolytic enzymes with a decrease in pH, with the most marked increases in binding occurring with phosphofructokinase, glyceraldehydephosphate dehydrogenase, enolase and pyruvate kinase. The significance of this data has been discussed with reference to the relative affinities of interaction of the individual glycolytic components and the physiological correlations of these phenomena.     

Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin

The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.     

Cytophotometry of post mortem glycogenolysis in different histochemical types of muscle fibres of the pig.

Synopsis Serial transverse sections of porcine longissimus dorsi muscle (18 pigs, 50 to 178 kg live weight) were reacted for NAD tetrazolium reductase and ATPase at pH9.4, and for glycogen with the periodic acid-Schiff (PAS) reaction. Three histochemical types of muscle fibre were identified; (1) strong ATPase and weak NADH oxidative activity; (2) strong ATPase and intermediate NADH oxidative activity; and (3) weak ATPase and strong NADH oxidative activity. Immediatepost mortem samples from one side of each animal were compared with a laterpost mortem sample from the other side by measuring the absorbance of PAS-stained glycogen at 570 nm with a microscope photometer. Laterpost mortem absorbance was expressed as a percentage of immediatepost mortem absorbance in each category of muscle fibre in order to compensate for distributional error and different starting levels of glycogen. Muscle fibres with weak ATPase and strong NADH oxidative activity showed a progressive decrease in absorbance of PAS-stained glycogenpost mortem. In some animals, fibres with strong ATPase and intermediate or weak NADH oxidative activity showed an initialpost mortem increase in absorbance of PAS-stained glycogen which was then followed by a progressive decrease. The maximum rates of decrease in absorbance in the three fibre types did not differ to any great extent.     

Effect of electrical stimulation on intracellular triacylglycerol in isolated skeletal muscle

The contribution of intracellular triacylglycerol (TG) as a substrate for skeletal muscle during electrical stimulation is equivocal. Therefore, the purpose of this study was to investigate the effect of electrical stimulation on the TG content in the isolated intact rat flexor digitorum brevis skeletal muscle preparation by use of two different stimulation protocols. Muscles were electrically stimulated for 1 h either continuously at 1 Hz or intermittently (30 s on, 60 s off) at 5 Hz while incubated in 21 degrees C Krebs bicarbonate buffer (pH 7.4) that contained 11 mM glucose. Control muscles were either frozen immediately after excision or incubated for 1 h. TG content was significantly decreased (P less than 0.05) compared with control concentrations in both stimulated muscle groups, with the greatest reduction (60%) occurring after 5-Hz intermittent stimulation. These data indicate that intramuscular TG is hydrolyzed in response to electrical stimulation in the isolated flexor digitorum brevis muscle preparation. In addition, the type of stimulation (higher frequency intermittent vs. lower frequency continuous) employed influences the amount of intracellular TG hydrolyzed.     

Cytophotometry of post mortem glycogenolysis in quiescent bovine muscle fibres in relation to temperature, succinate dehydrogenase activity and adenosine triphosphatase activity

Summary Immediatepost mortem samples of sternomandibularis muscles from six steers were maintained with a minimum of intrinsic activity at approximately 40°C or allowed to cool to approximately 22°C. Samples were frozen in liquid nitrogen at 0, 2, 4 and 6hpost mortem and serial transverse sections were stained by the periodic acid-Schiff (PAS) reaction for glycogen or reacted for adenosine triphosphatase (ATPase) or succinate dehydrogenase. Individual fibres were mapped and categorized from their ATPase and succinate dehydrogenase activity using a projecting microscope. The absorbance of PAS-stained glycogen in individual fibres was measured with a microscope photometer at 570 and 601 nm with a correction for distributional error. Overall, thepost mortem decline in absorbance was approximately twice as fast in body temperature samples relative to room temperature samples. Transientpost mortem increases in absorbance were detected in some situations, particularly in fibres with strong ATPase activity from room temperature samples. In fibres with strong ATPase activity, the rate of decline in absorbance increased progressivelypost mortem. Fibres with weak ATPase mostly had a lower initialpost mortem absorbance and were generally the first to become PAS-negative.     

The influence of molecular crowding on the binding of glycolytic enzymes to cytoskeletal structure

In a study of the interactions between glycolytic enzymes and cytoskeletal structure, the effect of increasing the degree of molecular crowding by the addition of protein was studied. This treatment resulted in an increased degree of binding of all the glycolytic enzymes, but with the most marked increases occurring with phosphofructokinase, enolase and pyruvate kinase. The significance of this data has been discussed in relation to the relative affinities of interaction of the individual components, the influence of molecular crowding and the physiological significance of this phenomenon.     

The spin-lattice relaxation times of water associated with early post mortem changes in skeletal muscle.

We studied the spin-echo signal of muscle water in a large time domain and found that the motion of the nuclear magnetic moment of tissue water cannot be characterized by a single spin-lattice relaxation time (T1). The relaxation time T1B, which is the T1 characterized by those protons with a slower relaxation rate, is influenced by the early post mortem changes in skeletal muscle. T1B increased with time after the tissue was taken from the animal and reached a maximum at 3 h. However, the weighted average of T1 of all water protons (T1A) did not change throughout the time course of the experiments.     

Functional diversity of the phosphoglucomutase superfamily: structural implications.

Three-dimensional structural models of three members of the phosphoglucomutase (PGM) superfamily, parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase, were constructed by homology modeling based on the known crystal structure of rabbit muscle phosphoglucomutase. Parafusin, phosphoglucomutase-related protein and sarcoplasmic reticulum phosphoglucomutase each have 50% or more identity with rabbit muscle phosphoglucomutase at the amino acid level and all are reported to exhibit no or minor phosphoglucomutase activity. There are four major insertions and two deletions in the parafusin sequence relative to PGM, all of which are located in surface-exposed loops connecting secondary structural elements. The remaining amino acid substitutions are distributed throughout the sequence and are not predicted to alter the polypeptide fold. Parafusin contains a putative protein kinase C site located on a surface loop in domain II that is not present in the homologs. Although the general domain structure and the active site of rabbit muscle phosphoglucomutase are preserved in the model of phosphoglucomutase-related protein, a major structural difference is likely to occur in domain 1 due to the absence of 55 amino acid residues in PGM-RP. This deletion predicts the loss of three alpha-helices and one beta-strand from an anti-parallel beta-sheet in this domain as compared with the rabbit muscle phosphoglucomutase.