A wire electrode arrangement for a horizontal electroblotting apparatus is described. The electrodes were mounted in electrode conduits built on perforated plexiglass plates. Electroblotting was ...carried out in a buffer tank. Human serum proteins were separated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes. Gas bubbles developed by electrolysis at the lower electrode were trapped in electrode conduits and removed by circulating buffer. Accumulation of gas bubbles between the electrodes during operation was avoided.
The effect of Aroclor 1248 (PCB) on the hepatopancreatic microsomal enzymes of carp was examined. The carp were fed a diet containing 250 ppm PCB for 7 days, and activities of NADPH-cytochrome c ...reductase, NADH-cytochrome b5 reductase, aminopyrine N-demethylase, and aryl hydrocarbon hydroxylase, along with the amount of cytochrome P-450, cytochrome b5, and microsomal protein increased significantly by ingestion of PCB. These results showed that Aroclor 1248 induced hepatopancreatic drug-metabolizing enzymes of carp which are similar to hepatic enzymes in mammals. The induction, however, seemed to be a mixed type, also induced by phenobarbital and 3-methylcholanthrene. By measuring the substrate-binding difference spectra of hepatopancreatic microsomes from the carp pretreated with Aroclor 1248, the PCB was found to be bound to cytochrome P-450 in a "modified type II" pattern as classified by Schenkman and others. The spectral changes induced by hexobarbital and aniline, and an increase in the δ-aminolevulinic acid synthetase activity also suggested that Aroclor 1248 increased the synthesis of cytochrome P-450 in the carp hepatopancreas. SDS-polyacrylamide gel electrophoresis, further demonstrated that the hepatopancreatic microsomes contained four kinds of heme protein and that the synthesis of the heme was induced by treatment of the carp with Aroclor 1248.
Pretreatment of rats with 3, 4, 5, 3', 4'-pentachlorobiphenyl (PenCB) at a single i.p. dose of 10 mg/kg caused a marked increase of cytochrome P-448 (P448) in liver microsomes. The liver microsomes ...of PenCB-treated rats (PenCB-microsomes) showed similar but somewhat different properties from those of 3-methylcholanthrene (MC)-treated rats (MC-microsomes) with respect to its CO difference spectrum, electrophoretic pattern on sodium dodecyl sulfate (SDS)-polyacrylamide gel and immunochemical reactivity with the antibodies prepared against the hemoproteins from phenobarbital (PB)- or β-naphthoflavone (BNF)-treated rat liver microsomes. Furthermore, PenCB pretreatment decreased not only demethylation of aminopyrine and codeine, but also hydroxylation of aniline, to which PenCB-microsomes showed lower affinity than MC-microsomes. Trace amounts of PenCB inhibited the metabolism of aniline and aminopyrine by MC-microsomes but not by untreated rat liver microsomes. On SDS-polyacrylamide gel electrophoretograms the ratio of cytochrome P450 (P450) to P448 in PenCB-microsomes was much smaller than that in MC-microsomes. These results may suggest that a part of the differences between the catalytic properties of PenCB- and MC-microsomes is due to the inhibitory effect of residual PenCB in PenCB-microsomes or/and the marked change of a relative ratio of P450 to P448 in PenCB-microsomes. A possibility that PenCB may induce a different P448 from that induced by MC is also discussed.
1. Changes of structural proteins in experimental and human myocardial infarction were studied by the determination of myosin- and acto-myosin-ATPase activities and gel electrophoretic analysis in ...the presence of sodium dodecyl sulfate (SDS). 2. In animal experiments using dogs, the relative amounts of myosin and α-actinin decreased at 24 to 48 hours after coronary ligation, became lowest at 72 hours, and remained at this level for 2 weeks and returned to almost normal value at 28 days. 3. Myosin-and actomyosin-ATPase activities decreased rapidly during 24 to 48 hours after ligation with temporary increase in their activities in the initial stage of ischemia and followed the similar time course as that of the amounts of myosin and α-actinin. 4. SDS gel electrophoretic analysis of structural proteins of infarcted tissues of the human hearts obtained from 5 cadavers showed also marked decrease of the contents of myosin and α-actinin with relative preservation of actin, tropomyosin and troponin-T.
1. Gel electrophoretic analysis of structural proteins extracted from myocardial tissues in the presence of sodium dodecylsulfate (SDS) is applicable to study the relative amount of structural ...proteins from less than 1Gm of heart tissue. 2. The compositions of structural proteins of the right and left atria and ventricles of the normal canine heart were identical and similar to those of skeletal muscle. 3. The compositions of structural proteins of the atrium and ventricle of the normal human heart were also identical.
1. Influence of ischemia on the biochemical properties of the sarcoplasmic reticulum (SR) was studied in the experimental myocardial infarction in the dog. 2. Ca2+-uptake rate of SR decreased at ...around 90 minutes after coronary occlusion. This reduction was roughly in parallel with the reduction in the Ca2+-Mg2+-stimulated ATPase activity. However, Ca2+-binding rate of SR was kept within the range of that of the non-infarcted tissue through the time course of myocardial infarction. 3. Ca2+-Mg2+-stimulated ATPase activity decreased at around 3 hours after coronary occlusion to about 50% of that of the non-infarcted portion. 4. In SDS gel electrophoresis, the protein band with the largest molecular weight amount three major components decreased at 3 hours after coronary occlusion, which is suggestive of ATPase. At 48 hours after coronary occlusion, the protein with the smallest molecular weight in the major proteins also decreased. 5. Ca2+-uptake rate, Ca2+-Mg2+-stimulated ATPase activity and the substructural changes returned to the normal level and pattern at around 28 days after coronary occlusion.