Herbert O. Hultin

Effect of NaCl on catalysis of lipid oxidation by the soluble fraction of fish muscle

Sodium chloride stimulated catalysis of oxidation of phosphatidylcholine liposomes by the soluble fraction of mackerel muscle. Chloride was determined to be the active component of the salt in this system. Sulfate also stimulated lipid oxidation. No difference was observed with either anion among sodium, potassium, or lithium cations. Redox iron was involved in the chloride stimulation of lipid oxidation by the press juice. Part of the chloride stimulation of the press juice was mediated through the high molecular weight (greater than 5 kdalton) fraction. Chloride improved the pro-oxidative effect of ascorbate on rat liver ferritin in vitro. It did not appear that production of chlorine radical by peroxidase was involved in the stimulatory effect of chloride.

Impact of citric acid on the tenderness, microstructure and oxidative stability of beef muscle

Acidification of meat can improve texture however it also increases susceptibility to lipid oxidation. The effect of injection and marination of citric acid to acidify and sodium carbonate or sodium tri-polyphosphate to increase pH of beef on tenderness, microstructure and oxidative stability was determined. Water-holding capacity and tenderness of beef semitendinosus muscle increased significantly at pH 3.52 upon addition of citric acid and returned to the level of untreated sample after pH was increased (pH ∼5.26) by sodium tri-polyphosphate. The microstructure of the muscle was lost upon acidification but reformed upon increasing muscle pH. Lipid oxidation was inhibited in cooked beef blocks and ground muscle acidified with citric acid. Lipid oxidation was also inhibited in citric acid acidified beef that was readjustment to pH values equal to or greater than the raw beef muscle with sodium tri-polyphosphate or sodium carbonate. In addition, citric acid that was adjusted to the pH of the raw beef so that it did not alter the pH of the beef also inhibited lipid oxidation. These results indicate that citric acid and not sodium tri-polyphosphate or pH adjustment was responsible for inhibiting lipid oxidation in beef. These results suggest that the best acid marination technique for beef would be citric acid since it is effective at both improving texture and inhibiting lipid oxidation.

Effect of deglycosylation on the stability of Aspergillus niger catalase

Abstract Catalase from Aspergillus niger was isolated from a commercial preparation. The enzyme was found to be glycoprotein containing 9% neutral sugar and 3% hexosamine. A. niger catalase was markedly more stable than the beef liver enzyme to inactivation by heat, proteolysis, and glutaraldehyde modification. The effect of glycosylation on thermal and proteolytic stability was investigated. Deglycosylation of the fungal enzyme resulted in increased susceptibility to proteolysis and a small decrease in thermostability.

Lipid peroxidation in fish muscle microsomes in the frozen state

Abstract The microsomal fraction from fish muscle has previously been shown to catalyze the oxidation of its lipid. In this study we have studied the rate of the reaction in the frozen state. The rate was dependent on temperature, decreasing with decreasing temperature. When the microsomes were frozen in the presence of NaCl there was greater activity than when they were frozen in the presence of KCl. The specific activity of the oxidation decreased with increasing protein concentration. This is possibly due to the limitation of oxygen in the frozen system. Lipid oxidation is a complex reaction and both initial products (lipid hydroperoxides) and breakdown products (those reacting with malondialdehyde) were measured. This ratio was relatively constant over a variety of conditions indicating that the rate-limiting step of the reaction occurred prior to the formation of lipid hydroperoxide. A study of the reaction at above-freezing temperatures and below-freezing temperatures in the presence of miscible solvents to prevent freezing at temperatures below 0 °C gave results which were consistent with the hypothesis that ice crystal formation had an accelerating effect on the reaction. Presumably this is due to concentration of reactants since freezing and thawing of the microsomes did not affect their rates of lipid oxidation. Potent inhibitors of the lipid oxidation reaction were found in the soluble fraction of the muscle tissue. These were both high-molecular and low-molecular-weight compounds. The low-molecular-weight inhibitors were more effective in the frozen state while the high-molecular-weight compounds were relatively more effective in the reaction catalyzed at temperatures above freezing.

Substrate inhibition of soluble and bound lactate dehydrogenase (isoenzyme 5).

Abstract Substrate inhibition of chicken lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5, was studied with the enzyme in the soluble phase and bound to muscle subcellular particulate structures. Inhibition studies were performed by incubating bound or soluble enzyme with NAD+ prior to measuring the reaction with a stopped-flow technique at 40 °C and a concentration of enzyme of 10−7 m . The value of V for soluble lactate dehydrogenase was 610 nmoles per sec, and for the bound enzyme it was 262. km (pyruvate) values were similar for both enzymes. Under our experimental conditions, up to 73% inhibition of the soluble enzyme was observed. On the other hand, there was no detectable inhibition of bound lactate dehydrogenase. It is suggested that the resistance to substrate inhibition of bound lactate dehydrogenase may possibly be due to the prevention of dissociation of the enzyme into monomeric or other subunits because of attachment to the particulate structures.

Fish muscle microsomes catalyze the conversion of trimethylamine oxide to dimethylamine and formaldehyde

The N-oxides of aliphatic amines are widely distributed in biological systems, but little is known about their function, synthesis, or degradation. In mammalian systems, the microsomal fraction of liver cells is the site of an oxidative dealkylation of tertiary amines requiring molecular oxygen and NADPH. Liver homogenates oxidize tertiary amines to amine oxides and cleave the resultant N-oxides to secondary amines and aldehydes [ 11. Marine teleosts and elasmobranchs contain high concentrations of trimethylamine oxide (TMAO) in their tissues which is thought to play an osmoregulatory role in these organisms [2] although this has been questioned [3]. The biosynthetic and biodegradative pathways of TMAO have been the subject of much controversy. The mechanism of TMAO breakdown in marine fish muscle has practical consequences since the process is accelerated during frozen storage [4], and the formaldehyde produced is thought to crosslink the fish muscle protein leading to a rapid toughening of the texture of some species, particularly gadoids [S]. Both enzymic and non-enzymic processes have been suggested as important in this process [6,7]. Enzymes catalyzing the breakdown of TMAO to dimethylamine and formaldehyde have been reported from fish organs such as the pyloric caeca, kidney and liver [6,8,9]. We report the isolation of a microsomal fraction from the skeletal muscle of red hake (Urophycis chuss) which causes a rapid breakdown of TMAO to dimethylamine and formaldehyde in vitro.

Factors affecting the distribution of lactate dehydrogenase between particulate and nonparticulate fractions of homogenized skeletal muscle

Abstract The distribution of lactate dehydrogenase between the particulate and soluble fractions of homogenized chicken breast muscle has been determined after centrifugation of the homogenate at 40,000 rpm for 30 minutes. The following factors were examined to determine their effect on the distribution: time before homogenization, time before centrifugation of the homogenized muscle, time of homogenization, pH and ionic strength of the homogenizing media, tissue concentration, and concentration of sucrose in the homogenizing media. The amount of lactate dehydrogenase released into the soluble fraction increased with increasing pH and ionic strength. An increase of solubility of the enzyme was also observed with increased tissue concentration; this is most likely related to the increase in ionic strength in the homogenate brought about by the extra tissue.

Influence of histidine on lipid peroxidation in sarcoplasmic reticulum

The free amino acid, histidine, which exists at high concentrations in some muscle systems, has previously been demonstrated to both inhibit and activate lipid peroxidation in membrane model systems. This study sought to characterize the specificity of histidines effect on iron-catalyzed enzymatic and nonenzymatic lipid peroxidation. Under conditions of activation (histidine added to the reaction mixture after ADP and ferric ion), alpha-amino, carboxylate, and pyrrole nitrogen were demonstrated to be involved by kinetic techniques in the activation of the enzymatic system. It is hypothesized that a mixed ligand complex (iron, ADP, and histidine) formed may allow rapid redox cycling of iron. While increasing concentrations of histidine led to increasing levels of stimulation in the enzymatic system, the maximum stimulation of a nonenzymatic lipid peroxidation system of ascorbate and ferric ion occurred at histidine concentrations near 2.5 mM. Inhibition of a nonenzymatic system (ferrous ion), on the other hand, occurred at all concentrations of histidine when the ferrous ion was exposed to ADP prior to histidine. In enzymatic systems, under conditions when the ferric ion was exposed to histidine prior to ADP, inhibition of lipid peroxidation by histidine also occurred. The inhibitory effect of histidine was ascribed to the imidazole group and may arise from the formation of a different iron complex or the acceleration of polymerization, dehydration, and insolubilization of the ferric ion by the imidazole nitrogen. The demonstrated ability of histidine to affect in vitro lipid peroxidation systems raises the possibility that this free amino acid may modulate lipid peroxidation in vivo.

Inhibition of enzymic and non-enzymic lipid peroxidation of flounder muscle sarcoplasmic reticulum by pretreatment with phospholipase A2

Sarcoplasmic reticulum, isolated from the muscle of winter flounder (Pseudopleuronectes americanus), was preincubated with phospholipase A2 to determine the effects of enzymic and non-enzymic lipid-peroxidation systems. Eicosapentaenoic acid (20:5) was preferentially released by phospholipase A2, but the percentage of docosahexaenoic acid (22:6) in the free fatty acids was similar to the percentage in membrane total fatty acids. A decrease in the production of both thiobarbituric acid-reactive substances and lipid hydroperoxides was observed in both the enzymic and non-enzymic peroxidation systems upon preincubation with phospholipase A2. Addition of palmitic acid or lysophosphatidylcholine did not inhibit either peroxidation system. Non-enzymic peroxidation was inhibited by the addition of beta-glycerophosphate, but enzymic peroxidation was apparently unaffected. A model is proposed to explain the inhibitory effect of phospholipid hydrolysis on lipid peroxidation in both systems, suggesting that fatty acids are structurally realigned upon hydrolysis, leading to decreased free-radical chain propagation. Additional factors appear to contribute to inhibition of enzymic peroxidation.

On the occurrence of multiple molecular forms of pectinesterase

Abstract Evidence is presented based on differential extraction procedures, pH-dependence studies, activation or inactivation by sodium dodecyl sulfate, and differential temperature inactivation that the pulp of the banana, Musa sapientum , contains at least three molecular forms of pectinesterase. Fraction I is extracted with water, Fraction II with 0.15 M NaCl, and Fraction III with 0.15 M NaCl at an alkaline pH. The pH-activity curves of the three fractions are considerably different. Fraction III retains a high percentage of its maximum activity at the pH of homogenized banana pulp, Fraction II retains much less, and Fraction I has very little activity at this pH. The effect of the anionic detergent, sodium dodecyl sulfate, on banana pectinesterase depends on the enzyme fraction assayed, on the pH at which the detergent is added, and, in the case of Fraction I, on whether the detergent is added to the enzyme solution before or after the pectin. Fraction I is much less heat-stable than Fractions II and III at pH 3.6 in the presence of substrate.