The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Comparative effects of experimental lead poisoning on enzymatic activities of kidney and liver in rat.

Rats were fed for 6 months with 0.1% lead acetate diet. At the end of the intoxication period urinary levels of ALA and lead were comparable with the usual urinary levels of chronically lead exposed humans. In kidney homogenates of poisoned rats succinate dehydrogenase, cytochrome oxidase and lactate dehydrogenase were unmodified, malate dehydrogenase and glutamate dehydrogenase were decreased, while Dt diaphorase and glucose-6-phosphate dehydrogenase were greatly increased compared with controls. In liver homogenates of the same poisoned animals no alteration of the enzymatic activities was observed.     

Histochemical studies on the adrenal glands of the marmosets (Callithrix jacchus and Callithrix penicillata).

The histrochemistry of the adrenal glands was studied in four adult male marmosets (two Callithrix jacchus and two Callithrix penicillata). It was impossible to demonstrate any reactivity to UDPG-GT, ADH, alanyl aminopeptidase, leucine aminopeptidase, xilitol (NAD-dependent) dehydrogenase, beta-glucuronidase and aryl-sulfatase in these glands. Total phosphorylase was found in scattered cells of the glomerulosa and adjacent outer fasciculata of one C. penicillata. The dehydrogenases (LDH, G-6-PDH,6-PGDH, NADPH2-TR,ICDH,SDH,NADH2-TR, alpha-GPDH, beta-OHBDH) as well as the hydrolases (except alkaline phosphatase, ATPase, and acetylcholinesterase) showed a stonger reactivity in the cortical part. Some hydrolases (naphthol acetate esterase, acid phosphatase) and cytochrome oxidase were less reactive in the zona glomerulosa, where the dehydrogenases were more abundant. The outer fasciculata and the reticularis also showed a strong dehydrogenase reactivity.     

Enzyme histochemical studies on rat lungs 2. Normal enzyme distribution in the alveolar tissue of the mature lung

Synopsis The activity and distribution of the following eighteen oxidative and hydrolytic enzyme systems have been investigated in the lung of the adult rat: reduced NAD dehydrogenase, reduced NADP dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, acid phosphatase, alkaline phosphatase, glucose-6-phosphatase, adenosine triphosphatase, 5-nucleotidase, non-specific esterase, cytochrome oxidase and -glucuronidase.The low concentration of cells in sections of inflated lung may have made histochemical demonstration of some enzymes impossible because the enzyme concentration was below that detectable by the method employed.The carboxylic acid cycle and the hexose monophosphate shunt were potentially active but fatty acid metabolism was not indicated.The granular reaction sometimes encountered in alveolar cell cytoplasm may be useful for differentiating alveolar cell types, but further cytochemical studies are required to resolve the possible metabolic differences of alveolar cells.     

Histochemical studies on the olfactory glomeruli of squirrel monkey

Summary Detailed histochemical studies on the distribution of various oxidative and dephosphorylating groups of enzymes have been made in the olfactory glomeruli of the squirrel monkey. The olfactory glomeruli showed strongly positive activity for succinic dehydrogenase, lactic dehydrogenase, monoamine oxidase, alkaline phosphatase, adenosinetriphosphatase and simple esterase. They showed moderately positive activity for cytochrome oxidase, specific cholinesterase, 5'nucleotidase; mildly positive activity for acid phosphatase; and negligible activity for nonspecific cholinesterase and glucose-6-phosphatase. The glomeruli did not show the presence of any thiamine pyrophosphatase-positive Golgi apparatus. The blood vessels surrounding the glomeruli were strongly positive for the nonspecific cholinesterase test. The significance of these results are discussed briefly.     

Pathways of glucose catabolism in procyclic Trypanosoma congolense

Studies of respiration on glucose in procyclic Trypanosoma congolense in the presence of rotenone, antimycin, cyanide, salicylhydroxamic acid and malonate have indicated the presence of NADH dehydrogenase, cytochrome b-c1, cytochrome aa3, trypanosome alternate oxidase and NADH fumarate reductase/succinate dehydrogenase pathway that contributes electrons to coenzyme Q of the respiratory chain. The rotenone sensitive NADH dehydrogenase, the trypanosome alternate oxidase, and cytochrome aa3 accounted for 24.5 +/- 6.5, 36.2 +/- 4.2 and 54.1 +/- 5.5% respectively of the total respiration. Activities of lactate dehydrogenase, NAD(+)-linked malic enzyme and pyruvate kinase were less than 6 nanomoles/min/mg protein suggesting that they play a minor role in energy metabolism of the parasite. Phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, succinate dehydrogenase, NADP(+)-linked malic enzyme, NADH fumarate reductase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase and glycerol kinase on the other hand had specific activities greater than 60 nanomoles/min/mg protein. These enzyme activities could account for the production of pyruvate, acetate, succinate and glycerol. The results further show that the amount of glycerol produced was 35-48% of the combined total of pyruvate, acetate and succinate produced. It is apparent that some of the glycerol 3-phosphate produced in glycolysis in the presence of salicylhydroxamic acid is dephosphorylated to form glycerol while the rest is oxidised via cytochrome aa3 to form acetate, succinate and pyruvate.     

Histochemical observations on the exoerythrocytic malaria parasite Plasmodium berghei in rat liver

Enzyme histochemical methods were performed on sporozoite infected liver tissue of rats in order to gain insight into the nutrition and metabolism of exoerythrocytic forms of Plasmodium berghei. The following enzymes were demonstrated in the hepatocytic stages of the parasites, obtained 41 and 48 h after inoculation of sporozoites: acid phosphatase, cytochrome oxidase, NADH-tetrazolium reductase, succinate dehydrogenase, NAD+ and NADP+ dependent isocitrate dehydrogenase, NADP+-dependent malate dehydrogenase, lactate dehydrogenases, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenases and alpha-glycerol-phosphate dehydrogenase. The results suggest that a conventional Embden-Meyerhoff pathway, pentose phosphate pathway and Krebs' citric acid cycle may in part be present in these exoerythrocytic parasites. Alkaline phosphatase, nucleoside polyphosphatase, 5' nucleotidase, glucose-6-phosphatase, alpha-glucan phosphorylase, NAD+ dependent malate dehydrogenase, amino-peptidase M and non-specific esterases were not detected by our techniques in the parasite. The enzyme distribution of this intrahepatocytic malaria parasite revealed by histochemistry is compared with the enzyme distribution in the other phases of the parasite's life cycle.     

Influence of oxygen tension on the respiratory activity of Mycobacterium phlei

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.     

Oxidative enzymes in the sebaceous glands of the domestic cat

Synopsis The distribution and activities of several oxidative enzymes in various regions of the sebaceous glands of the domestic cat have been studied. The results obtained emphasize the outstanding importance of NADP-linked dehydrogenases for lipogenesis during sebum production. In particular, the reactions for glucose-6-phosphate dehydrogenase were very strong. Among the NAD-linked dehydrogenases investigated, lactate dehydrogenase showed strong activity in the peripheral cells of the sebaceous gland. The reactions for cytochrome oxidase and succinate dehydrogenase were weaker.     

Enzymatic characterization of the pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP)

Glucose is the main physiological stimulus for insulin biosynthesis and secretion by pancreatic beta-cells. Glucose-6-phosphatase (G-6-Pase) catalyzes the dephosphorylation of glucose-6-phosphate to glucose, an opposite process to glucose utilization. G-6-Pase activity in pancreatic islets could therefore be an important factor in the control of glucose metabolism and, consequently, of glucose-dependent insulin secretion. While G-6-Pase activity has been shown to be present in pancreatic islets, the gene responsible for this activity has not been conclusively identified. A homolog of liver glucose-6-phosphatase (LG-6-Pase) specifically expressed in islets was described earlier; however, the authors could not demonstrate enzymatic activity for this protein. Here we present evidence that the previously identified islet-specific glucose-6-phosphatase-related protein (IGRP) is indeed the major islet glucose-6-phosphatase. IGRP overexpressed in insect cells possesses enzymatic activity comparable to the previously described G-6-Pase activity in islets. The K(m) and V(max) values determined using glucose-6-phosphate as the substrate were 0.45 mm and 32 nmol/mg/min by malachite green assay, and 0.29 mm and 77 nmol/mg/min by glucose oxidase/peroxidase coupling assay, respectively. High-throughput screening of a small molecule library led to the identification of an active compound that specifically inhibits IGRP enzymatic activity. Interestingly, this inhibitor did not affect LG-6-Pase activity, while conversely LG-6-Pase inhibitors did not affect IGRP activity. These data demonstrate that IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors to this molecule can be obtained. IGRP inhibitors may be an attractive new approach for the treatment of insulin secretion defects in type 2 diabetes.     

Subcellular localisation of guinea pig hepatic molybdenum hydroxylases.

Molybdenum hydroxylase activity in guinea pig liver has been compared with that of marker enzymes in mitochondria (succinate dehydrogenase), microsomes (glucose-6-phosphatase) and cytosol (lactate dehydrogenase). Aldehyde oxidase activity was highest in the cytosol, with about 10-fold activity of xanthine oxidase. Significant molybdenum hydroxylase activity was found in mitochondria with minimal levels in microsomes. Mitochondrial and cytosolic aldehyde oxidase varied in substrate specificity and electrophoretic mobility with two major bands in each fraction, one of which was common to cytosol and mitochondria.     

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.     

Acid hydrolases in HeLa cells: comparison of methods for light microscopy

To distinguish lysosome populations of HeLa cells, acid phosphatase, beta-glucuronidase, arylsulfatase and esterase were demonstrated using various substrates and couplers with different fixations, pHs and inhibitors. The substrates chosen were for acid phosphatase, naphthol AS-BI phosphate with fast red violet LB at pH 4.6; for beta-glucuronidase, naphthol AS-BI beta-D-glucuronide with fast red violet LB at pH 4.4; for arylsulfatase, p-nitrocatechol sulfate, with lead as the capturing ion, at pH 4.8 and 5.6; and for esterase, naphthol AS-D acetate with fast blue BB at pH 6.5. In the azo-dye methods, the coupling was always simultaneous and results were satisfactory with unfixed cells. For optimal demonstration of arylsulfatase, cells were fixed in glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, 2% for 24 hr or 6.25% for 2 hr, and washed for 1-9 days in 0.1 M veronal acetate buffer pH 7.2, 7.5% with respect to sucrose. Two groups of lysosomes were distinguished. One comprised small bodies, probably primary lysosomes, which lay in a cluster near the nucleus. They had quite stable membranes and were mostly acid phosphatase-positive. They sometimes contained beta-glucuronidase or esterase, but rarely arylsulfatase. The other group included all the acid hydrolase-positive bodies scattered throughout the rest of the cytoplasm. They were mostly larger, with more labile membranes, and contained beta-glucuronidase, esterase or arylsulfatase, but rarely acid phosphatase.     

Isolation and characterization of cardiac sarcolemma.

A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K+-stimulated p-nitrophenylphosphatase, (Na+ + K+)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca2+-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca2+ binding).     

Studies on cartilage formation. XX. Histochemical investigation of some enzymes of glycogen metabolsim in regenerative articular surfaces.

In 28 dogs the distal articular cartilage of the femur was removed and the regenerating articular surface on the 70th postoperative day was studied histochemically for hexokinase, glucose-6-phosphatase, phosphohexose-isomerase, fructose-1, 6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, lactate dehydrogenase isoenzymes, phosphoglucomutase, phosphorylase, glycogen synthetase, UDP--glucose dehydrogenase, and UDP-glucuronic acid-4-epimerase. The articular surface consisted of fibrous tissue and of cartilage islets. The latter contained cells differentiating into cartilage and young chondrocytes. The glycolytic enzymes reacted positively in the regenerative articular surface. Enzyme activities were higher in the cells (particularly the chondroblasts and young chondrocytes) of the cartilage islets than in the connective tissue. In the cells differentiations into cartilage, beside the LDH isoenzymes characteristic of glycolysis, a significant LDH1 and LDH2 activity was observed. At the same site the presence of fructose-1, 6-diphosphatase-activity could be assumed, but there was no glucose-6-phosphatase activity. Glycogen synthesis proceeded in the cells of the cartilage islets and UDP-glucuronic acid-4-epimerase activity was observed in the differentiated cells. UDP-glucose dehydrogenase activity was positive in every section of the articular surface.     

Salivary glands of the cat: A histochemical study

Synopsis The submandibular, sublingual and parotid glands of the cat have been studied. Mucosubstance histochemistry demonstrated acidic mucosubstances with varying properties in the acini. Thiamine pyrophosphatase and nucleoside diphosphatase reaction products were seen with a Golgi-like appearance in acinar cells. Granules of acid phosphatase, -glucuronidase and E600-resistant esterase reaction products, presumably representing lysosomal enzyme activities, were seen in acinar and ductal cells. Diffuse acid phosphatase and -glucuronidase reaction products were seen in central cells of the submandibular acini, and diffuse non-specific esterase reaction product was seen in acinar and ductal cells. Arylamidase reaction product was associated with some acinar cells. Reaction product from a peroxidase technique was seen in demilunar cells of the submandibular acini, in parts of the sublingual acini, in parotid acini, and in ductal cells. Cytochrome oxidase and succinate dehydrogenase reaction products were seen most strongly in striated ducts, whereas NADH- and NADPH-diaphorase reaction products were seen at a high level throughout the ducts.     

Comparative distribution of four lysosomal enzymes within various structures of the human placenta

The histochemical activity of 4 lysosomal enzymes, acid phosphatase (AP), nonspecific esterase (NE), aryl sulphatase (AS), and beta-glucuronidase (BG), was compared in following structures of the human placenta: syncytiotrophoblast, villous stromal cells, fetal capillaries and larger blood vessels, cells of basal plate, macrophages, and Hofbauer cells. In spite of a general similarity in distribution of the investigated enzymes, differences concerning particular structures were found. Thus positively stained granules in endothelia of capillary vessels were revealed only in the reaction for BG, although contours of capillaries were also outlined by the diffuse reaction product for AS. The muscular layer of larger vessels reacted strongly for AS and weakly for NE with the remaining reactions being negative. In syncytiotrophoblast, BG appeared much less active than the other 3 enzymes. The possible significance of the BG positive granules in endothelial of capillaries and of the occasional divergence in distribution of the classical lysosomal markers (AP and BG) is discussed.     

Localization of Acid Hydrolase Activity in Zea mays L. Root Tips

Naphthol AS-BI phosphatase, esterase, aryl sulphatase, glucuronidase,and ß-glycerophosphatase have been studied in frozensections of maize root tips. In general these enzymes showedhighest activities at the root surface and at particulate sitesin the cytoplasm although the indigogenic method for esteraseshowed no particulate activity and the naphthol AS-D acetatereaction gave no pronounced surface activity. With electronmicroscopy highest activity for ß-glycerophosphatasewas observed in the cell walls and associated with the vacuoles.The significance of these observations are discussed in relationto the function of surface hydrolytic activity and to the presenceof lysosome-like bodies in higher plant cells.     

Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.