A histochemical study of changes in mitochondrial enzyme activities of rat liver after ischemia in vitro

Changes in the activity of three mitochondrial enzymes in rat liver after in vitro ischemia have been determined by enzyme histochemical methods. The changes were correlated with the appearance in the electron microscope of flocculent densities in the mitochondria indicative of irreversible cell injury. The flocculent densities were observed in rat liver after about 2 h of ischemia in vitro at 37 degrees C. At the same time the activity of glutamate dehydrogenase, localized in the mitochondrial matrix, started to decrease. However, the activities of succinate dehydrogenase localized in the inner membrane of mitochondria, as well as monoamine oxidase of the mitochondrial outer membrane did not change at that stage. It is concluded from the results of this study and those of others that flocculent densities are formed by denaturation of proteins of the mitochondrial matrix in which glutamate dehydrogenase takes part. It should be considered more as a sign than as the cause of cell death.     

Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF- secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF- detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF- is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.     

Superoxide dismutase in liver ischemia

The properties of Cu,Zn-superoxide dismutase (SOD) from rat liver after 2-hour total ischemia or after ischemia with subsequent 24-hour reperfusion were studied. Two hours after ischemia the specific activity of SOD decreases drastically (about 3-fold) - from 510 +/- 11 u./mg in normal tissue and 196 +/- 33 u./mg after ischemia showing a further increase after reperfusion (276 +/- 40 u./mg). Using competitive immunoenzymatic analysis, the relative contents of SOD in the cytosol were determined. After ischemia the SOD content in the cytosolic fraction decreased (approximately 3-fold) but returned to the initial level after reperfusion. Polyacrylamide gel electrophoresis revealed that in control samples active SOD is heterogeneous and produces 3-4 bands, similar to the purified SOD from rat liver. After the ischemia the intensity of minor fast band IV increased and a new band V of a still higher mobility appeared. After the reperfusion the electrophoretic patterns were similar to control. Two or three times more SOD antigen from ischemia liver cytosol was absorbed to the surface of polystyrol plate in a direct sorption enzyme immunoassay procedure as compared to that from intact liver cytosol. It is suggested that the decreases of amount and the activity as well as changes of properties of SOD could be due to its oxidative modification and degradation of the modified enzyme.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O ₂ ^⋅ and H₂O₂ during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-β-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O₂ consumption, only slightly but substantially inhibited in a competitive manner the O ₂ ^⋅ -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive Superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria. These results are interpreted in terms of a possible lysosomal membrane permeability to O ₂ ^⋅ causing organelle impairment by a process that, though leading to enzyme-marker leakage, does not involve lipid peroxidation.     

TNF—α转换酶的结构特征及抑制剂

肿瘤环因子－α转换酶（ｔｕｍｏｒ ｎｒｃｒｏｓｉｓ ｆａｃｔｏｒ－α ｃｏｎｖｅ ｒｔｉｎｇ ｅｎｚｙｍｅ,ＴＡＣＥ）将２６ｋＤ膜结合型ＴＮＦ－α前体水解为具有生物活性的可溶性１７ｋＤ ＴＮＦ－α。ＴＡＣＥ基因克隆的成功,主宰其为金属水解蛋白（ａｄａｍａｌｙｓｉｎ）家族的膜结合型异整合素金属蛋白酶。发现许多金属蛋白酶抑制剂ｈｙｄｒｏｘａｍａｔｅ类化全物能抑制ＴＡＣＥ活性阻断ＴＮＦ－α释放,并保护脓毒     

Enzyme leakage and multipoint attachment of agarose-bound enzyme preparations

The solvolytic detachment of leucine aminopeptidase from Sepharose-enzyme conjugates with multiple and single anchoring bonds has been studied under a variety of conditions by radiochemical and enzymological methods. The release of the single-point-fixed conjugate could be described by a leakage function, derived previously, yielding the first-order rate constant of the cleavage of the enzyme-matrix bond. The nucleophile hydroxylamine increased the detachment rate considerably. The release of the immobilized enzyme was incomplete in all experiments even after prolonged times. The enzyme leakage from multipoint-attached conjugates was still high enough to prohibit a long-term use of such preparations in routine work at room temperature.     

Increased expression of peroxiredoxin 1 and identification of a novel lipid-metabolizing enzyme in the early phase of liver ischemia reperfusion injury

Warm ischemia reperfusion (IR) injury of the liver is associated with changes in the expression and/or post-translational modification of numerous proteins. Only a few of these have been identified. We used 2-D DIGE to identify cytosolic proteins altered in the early stage of IR in an established rat model of segmental hepatic ischemia. Proteins in 18 abundant spots altered by IR were identified by LC-MS/MS and Western blot. Many identified proteins were enzymes involved in glucose and lipid metabolism. Isoamyl acetate-hydrolysing esterase 1 homolog, not previously characterized in liver, was also identified. A threefold increase in peroxiredoxin 1 (Prx1) and its oxidized forms was observed as was an increase in Prx1 mRNA. Peroxiredoxins and their overoxidation have previously been associated with IR. In contrast to other studies, we did not detect typical overoxidation of Prx1 on the peroxidatic cysteine (Cys(52)). Instead, we identified novel overoxidation of the resolving cysteine (Cys(173)) residue by LC-MS/MS. Our results show that a rapid increase in Prx1 expression is associated with the early phase of IR of the liver, likely contributing to mechanisms that protect the liver against IR damage. Additionally, we have revealed a potential role in liver for a novel lipid-metabolizing enzyme.     

Cholinergic enzyme activity during partial brain ischemia in the dog

Activity of cholinacetyltransferase (ChAT. EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) was monitored during occlusion of arteria cerebri media dx. (MCA) in five areas of the brain cortex, in nucleus caudatus and in the thalamus of the ipsilateral and contralateral hemisphere. After 1 hour of MCA occlusion ChAT and AChE activity was reduced in the ischemised region of the hemisphere, i. e. in gyrus ectosylvius anterior and gyrus sylvius anterior, whereas after 4 hours of occlusion the differences were not significant. In nc. caudatus and thalamus the activity of enzymes during ischemia did not change much.