ABSTRACT
In a preceding article, we described alterations occurring in rat pancreas acinar cells at successive post-mortem (PM) intervals. In ultra-thin sections from samples obtained from 0.5, 1, 2, 4, 8 and 12 h, we observed in the Golgi apparatus the appearance of an anomalous membrane bound structure. Such structures are formed by tubules and vesicles that we have called tubular vesicular structure (TVS), and they are frequently located in the position corresponding to the 4th cisterna of the Golgian cisternal pile. Lobules of rat pancreas, incubated in vitro with metabolic inhibitors (such as antimycin A, sodium fluoride, sodium azide and potassium cyanide), were processed in order to be compared with the PM samples of the rat acinar cells. In sliced pieces of lobules, acid phosphatase (AcPase) and tiaminopirophosphatase (TPPase) activity were evaluated. Except for the potassium cyanide treatment, we frequently observed the TVS located at the position corresponding to the 4th cisternae (similar to those observed in the PM acinar cells). These TVS's are predominantly TPPase positive. Based on this result and the fact that the TVS's are surrounded by a membrane (as confirmed by the freeze-fracture replica results) with no structural elements inside, they seem not to correspond to autophagosomes. The TVS's, observed either at PM consecutive times or incubated with metabolic inhibitors, seem to be structures formed in response to ATP deprivation. In 0,5 h PM cells and in cells incubated for 30 and 60 min with metabolic inhibitors, the subcellular structures reacted for AcPase in the rigid lamellae, CV and lysosomes.
Subject(s)
Golgi Apparatus/enzymology , Membrane Transport Proteins/metabolism , Pancreas/enzymology , Acid Phosphatase/analysis , Acid Phosphatase/antagonists & inhibitors , Animals , Antimetabolites/pharmacology , Antimycin A/pharmacology , Enzyme Inhibitors/pharmacology , Freeze Fracturing , Golgi Apparatus/drug effects , Golgi Apparatus/ultrastructure , Histocytochemistry , In Vitro Techniques , Membrane Transport Proteins/drug effects , Pancreas/drug effects , Pancreas/ultrastructure , Potassium Cyanide/pharmacology , Protein Transport/drug effects , Protein Transport/physiology , Rats , Rats, Wistar , Sodium Azide/pharmacology , Sodium Fluoride/pharmacology , Thiamine Pyrophosphatase/analysis , Thiamine Pyrophosphatase/antagonists & inhibitors , Time FactorsABSTRACT
The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.
Subject(s)
Acid Anhydride Hydrolases , Golgi Apparatus/enzymology , Microsomes, Liver/enzymology , Phosphoric Monoester Hydrolases/analysis , Pyrophosphatases/analysis , Thiamine Pyrophosphatase/analysis , Adenosine Triphosphate/pharmacology , Animals , Hydrogen-Ion Concentration , Isoelectric Focusing , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/metabolism , Rats , Substrate Specificity , Thiamine Pyrophosphatase/antagonists & inhibitors , Thiamine Pyrophosphatase/metabolismSubject(s)
Alcohol Deterrents/pharmacology , Alcoholic Intoxication/enzymology , Catechin/pharmacology , Kidney Cortex/enzymology , Kidney Tubules, Proximal/enzymology , 5'-Nucleotidase , Acid Phosphatase/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Alkaline Phosphatase/antagonists & inhibitors , Animals , Enzyme Activation/drug effects , Female , Kidney Cortex/drug effects , Kidney Tubules, Proximal/drug effects , Male , Nucleotidases/antagonists & inhibitors , Rats , Thiamine Pyrophosphatase/antagonists & inhibitorsABSTRACT
Analysis of five brains from patients with Leigh's disease demonstrates an accumulation of thiamine pyrophosphate and a deficiency of thiamine triphosphate. The enzyme which converts thiamine pyrophosphate to thiamine triphosphate was normally active in two of these brains, suggesting that the inhibitor found in Leigh's disease is probably producing the observed neurochemical changes. Reasons for the histological similarity between Leigh's and Wernicke's diseases are suggested.
Subject(s)
Brain Chemistry , Encephalomalacia/metabolism , Pyrophosphatases/antagonists & inhibitors , Thiamine Pyrophosphatase/antagonists & inhibitors , Thiamine/analysis , Brain/enzymology , Brain Stem , Encephalomalacia/enzymology , Enzyme Inhibitors/analysis , Humans , Phosphates/analysis , Thiamine Pyrophosphatase/metabolism , Thiamine Pyrophosphate/analysisABSTRACT
The basic properties of microsomal thiamine diphosphatase (TDPase) in rat brain were examined. It was mainly localized in the microsomal fraction. Microsomal TDPase showed an absolute requirement for divalent cation which was best satisfied by Ca++. Theophylline (1 mM), NADP (0.1 mM) and NADPH (0.1 mM) significantly inhibited its activity. However, caffeine, theobromine, NAD and various putative neurotransmitters did not affect the enzyme activity. UDP was inhibitory and IDP was a competitive inhibitor, but ATP had no effect on the enzyme activity.
