Effects of Aldehyde Fixation on Adenosine Triphosphatase and Peroxidase Activities in Maize Root Tips

The effects of aldehyde fixation of excised roots and subcellularfractions of Zea mays have been studied in relation to the preservationof ATP-ase and peroxidase activities. Total peroxidase was littleaffected by either formaldehyde or glutaraldehyde whereas ATP-aseshowed considerable loss of activity, particularly with glutaraldehyde.The activity remaining after fixation was dependent on boththe concentration of fixative and the pH of fixation. Subcellularfractions differed in their response to fixation with the cellwall fraction generally showing higher retention of activitythan the mitochondrial or microsomal fractions.     

Cytochemical localization of myrosinase (β-thioglucosidase) in root tips ofSinapis alba

Summary A method for electron microscopic cytochemical localization of a-thioglucosidase (myrosinase) has been developed. Since sulphate is one of the products of the hydrolysis of sinigrin by myrosinase, it was felt that if the incubation was carried out in the presence of Pb⁺⁺-ions an insoluble precipitate of electron-dense PbSO₄ would be formed at the reaction sites. Following formaldehyde fixation a few different cell organelles in the extreme root tip ofSinapis alba showed reaction specificity for myrosinase but following glutaraldehyde fixation the enzymatic activity was inhibited. Biochemical tests of the isolated enzyme showed complete inhibition of the myrosinase by glutaraldehyde. Variations in the substrate concentration and incubation time indicated that the enzyme was confined to the dilated cisternae of the endoplasmic reticulum and in a limited extent to the mitochondria.     

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.     

Electroncytochemical and biochemical demonstration of guanylate cyclase activity in the pancreatic islet

Summary With a cytochemical method using guanylyl imidodiphosphate as a substrate, the guanylate cyclase activity was localized on the plasma membrane of A, B and D cells of islets of Langerhans isolated from the rat. Adequate control experiments were performed by a double-blind method. Parallel biochemical assay showed that guanylate cyclase activity was not completely lost after fixation with 1% glutaraldehyde and incubation with 4 mM lead nitrate. Furthermore, the depressed activity was still stimulatable with acetylcholine.This investigation was supported by grants from the Bickell Foundation and the Medical Research Council of Canada (MA-868). Part of this work was presented at the 35th Annual Meeting of the American Diabetes Association on June 16, 1975     

Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex.

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.     

Electroncytochemical and biochemical demonstration of guanylate cyclase activity in the pancreatic islet.

With a cytochemical method using guanylyl imidodiphosphate as a substrate, the guanylate cyclase activity was localized on the plasma membrane of A, B and D cells of islets of Langerhans isolated from the rat. Adequate control experiments were performed by a double-blind method. Parallel biochemical assay showed that guanylate cyclase activity was not completely lost after fixation with 1% glutaraldehyde and incubation with 4 mM lead nitrate. Furthermore, the depressed activity was still stimulatable with acetylcholine.     

Directed evolution of the surface chemistry of the reporter enzyme beta-glucuronidase.

The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.     

Ultracytochemical detection of nucleoside phosphatase activity in human peripheral blood cells

The authors elaborated and described the optimum conditions for fixation, incubation and preparation of human blood cell samples in minimum quantities for ultrastructural and ultracytochemical investigations of 5'-nucleotidase and ATPase activities. The best preservation of the blood cell ultrastructure was obtained after fixation with buffered 1% glutaraldehyde solution followed by postfixation in buffered 1% OsO4 solution. The best ultracytochemical demonstration of 5'-nucleotidase and ATPase activities was achieved after fixation in buffered 2% formaldehyde prior to cytochemical incubation. DMSO added to either fixation or incubation media was shown to damage the plasmalemma and glycocalyx structure in cell suspensions. ATPase in 5'-nucleotidase activities were revealed in plasmalemma, cytoplasmic reticulum, Golgi complex, mitochondria and in the nuclei, in particular, in the perinuclear space, nucleolus and chromatin. With respect to the localization and activity of nucleosidephosphatases, lymphocytes proved to be most heterogenic, with the enzyme activity level directly depending on the rate of ultrastructural differentiation in lymphocytes.     

Tissue fixation with diimidoesters as an alternative to aldehydes. II. Cytochemical and biochemical studies of rat liver fixed with dimethylsuberimidate.

Rat liver fixed with dimethylsuberimidate (DMS) was studied to investigate the use of diimidoesters as dixatives for light and electron microscopic cytochemistry. Paraffin sections of liver fixed with DMS at pH 9.5 were weakly stained with the ninhydrin-Schiff procedure, indicating extensive reaction of NH3+ groups with the fixative. Nuclei were strongly strained by the Feulgen procedure, with no backgroud reaction. In contrast, glutaraldehyde fixation resulted in a significant background reaction in the cytoplasm and nuclei in controls for the Schiff-based stains. DMS-fixed liver stained intensely for glycogen with the Periodic acid-Schiff procedure, and biochemical analysis of glycogen retention and extractability indicated that DMS retained considerably more glycogen in sections than glutaraldehyde. DMS-fixed liver incubated for thiamine pyrophosphatase activity revealed reaction product in ER cisternae, Goli saccules and bile canaliculi. Peroxisomes were strongly reactive for catalase activity after incubation in diaminobenzidine medium, and reaction product of glucose-6-phosphatase activity was considerably greater following DMS fixation than after glutaraldehyde. Biochemical studies revealed up to twice as musch residual activity of glucose-6-phosphatase after DMS fixation. These results suggest that DMS may be useful as a primary fixative for certain cytochemical procedures.     

Effect of fixation on demonstration of phosphatases of Eimeria tenella grown in chick kidney cell cultures.

The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase greater than thiamine pyrophosphatase greater than 5'-nucleotidase greater than adenosine triphosphatase greater than acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase, TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase. Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.     

Effect of Fixation on Demonstration of Phosphatases of Eimeria tenella Grown in Chick Kidney Cell Cultures

SYNOPSIS. The effects of fixation with various concentrations of glutaraldehyde or formaldehyde, acetone or ethanol, and freeze-drying on 5 phosphatases of Eimeria tenella and chick kidney cell cultures were demonstrated in situ. Gultaraldehyde inactivated the phosphatases more than did the formaldehyde, but the effect of the combination of the 2 (Karnovsky's fixative) was greater than that of either glutaraldehyde or formaldehyde alone. The higher the concentration of aldehyde and the longer the duration of exposure, the greater the inactivation. The order of sensitivity to aldehyde fixation of the enzymes tested was glucose-6-phosphatase > thiamine pyrophosphatase > 5'-nucleotidase > adenosine triphosphatase > acid phosphatase. Cytologic detail was preserved more efficiently with glutaraldehyde than with formaldehyde. Optimal preservation of enzyme activity for cytochemistry was with 2% glutaraldehyde for 30 min or 2% formaldehyde for 1 hr for G-6-Pase. TPPase, and 5'-nucleotidase, and with 2% glutaraldehyde or 2% formaldehyde for 2 hr with ATPase and AcPase.
Quenching with subsequent fixation in cold acetone or ethanol resulted in complete inactivation of G-6-Pase, TPPase, and 5'-nucleotidase; although cells fixed in this manner yielded large amounts of reaction product for ATPase and AcPase, the distribution was diffuse, and some of it appeared to be artifactual. Quenching with subsequent freeze-drying was unsatisfactory because nearly all of the cell layers rolled off the cover glasses.     

Cytochemistry of adenylate cyclase. Quantitative analysis of the effect of cytochemical procedures on adenylate cyclase in heart tissue homogenates

The effects of different preparative and cytochemical procedures on adenylate cyclase (AC) activity in heart muscle homogenates were studied by quantitative analysis. We were mainly concerned with perfusion prefixation, using glutaraldehyde (GA) with and without DMSO, and with the influence of cytochemical incubation with lead ions as the capture reagent. Furthermore, we measured the direct effect on the AC activity of lead, cobalt, and strontium ions in prefixed heart homogenates. We also studied the influence of phosphatidylinositol and 2',5'-dideoxyadenosine. The following results were obtained: 1. Perfusion fixation using 2% GA buffered with cacodylate reduced the AC activity by about 20%. After the entire cytochemical procedure was finished, 20% of the original AC activity was still present. Stimulation by epinephrine, histamine and fluoride, which increased the activity of AC two or three times in our experiments, was only slightly reduced by the cytochemical treatments. 2. Lead ions (2 mM), added to the biochemical assay without chelating compounds, reduced the AC activity about 90%. 5 muM phosphatidylinositol stabilized the fluoride- and hormone-sensitive AC activity. 3. Co2+ also reduced the AC activity, though less than Pb2+. Sr2+ had no effect on the basic activity of the AC but had a slightly inhibitory effect on the hormone and fluoride stimulation. 4. 5% DMSO added to the fixative had no influence on the basic activity of the AC. However, this solvent definitely reduced the level of stimulation by fluoride and guanine nucleotide plus hormones. 5. A potential inhibitor of enzyme activity and of the hormone- and fluoride-sensitive AC was the adenosine derivative, 2',5'-dideoxyadenosine. This compound, at a concentration of 10(-3) M, inhibited all AC activity in the heart homogenates.     

Cytochemical localization of plasma membrane ATPase activity in plant cells

Summary The cytochemical localization of ATPase activity has been investigated in maize root cells using both lead and cerium-based capture methods. With both methods, staining at the plasma membrane was observed in all cells of the root, although the precipitate obtained with cerium was more uniform and granular than that with lead. Controls using no substrate or no magnesium, -glycerophosphate to replace ATP, vanadate or boiled tissue generally showed little or no staining. However, biochemical studies on purified plasma membrane fractions showed that ATPase activity was markedly inhibited by fixation, particularly by glutaraldehyde, and also by lead and cerium ions. Non-enzymic hydrolysis of ATP by cerium was greater than that by lead. The value and limitations of these procedures for the localization of plasma membrane H⁺-ATPase activity are summarized in relation to previous criticisms of these methods.Abbreviations DTT dithiothreitol - EDTA ethylene diaminetetraacetic acid - GP B-glycerophosphate - PCMBS p-chloromercuribenzene sulphonic acid - PMSF phenylmethylsulphonyl fluoride     

Cytochemical demonstration of adenylate cyclase with strontium chloride in the rat pancreas

A cytochemical procedure for the localization of adenylate cyclase with Sr2+ as the capture ion and adenylyl imidodiphosphate as the specific substrate was evaluated in the rat pancreas. Incubation medium was unaffected by the addition of 5 mM strontium ions but became turbid in the presence of lead or strontium plus 10 mM NaF. Tissues were prefixed in 2% formaldehyde/0.5% glutaraldehyde and incubated, and the cytochemical precipitate was converted to the Pb2+ salt. Enzymatic activity was demonstrated on the plasma membrane of pancreatic acinar cells and responded to stimulation by secretin. Controls frequently contained Pb2+ sequestered in mitochondria, but otherwise only a few randomly distributed grains were observed. The controls were 1) omission of substrate from the medium; 2) incubation of tissue for 1 min in complete medium; and 3) tissue previously inactivated by microwave irradiation and incubated for 30 min in complete medium including secretin.     

Cytochemical Localization of K-stimulated Adenosine Triphosphatase Activity in Xylem Parenchyma Cells of Barley Roots

ATPase activity in xylem parenchyma cells of barley (Hordeum vulgare L.) roots was demonstrated cytochemically with a lead precipitation reaction. The methodical parameters of this cytochemical test were optimized for distinction between ATPase-specific and nonspecific precipitates. Optimum conditions were prefixation in 1% glutaraldehyde for 1 hour and incubation for 2 hours in a medium containing 2 mm each of ATP, Ca(2+), and Pb(2+) at pH 7 and 25 C. Problems of cytochemical localizations are discussed.ATPase activity occurred mainly at the plasmalemma, the endoplasmic reticulum nuclear envelope, and outer mitochondrial membranes of xylem parenchyma cells. The tonoplast of these cells showed only little ATPase activity. High K(+) concentrations stimulated ATPase activity, particularly at the plasmalemma. Diethylstilbestrol prevented the formation of ATPase-specific precipitates. The cytochemical demonstration of a K(+)-stimulated ATPase at the plasmalemma of xylem parenchyma cells is discussed in relation to the possible role of this membrane in ion transport to the vessels.     

Effect of glutaraldehyde and lead on the activity of hepatic glucose-6-phosphatase. A biochemical and cytochemical study.

The sensitivity of mouse liver glucose-6-phosphatase activity towards glutaraldehyde fixation has been analysed by biochemical and cytochemical means. The degree of enzymatic inhibition and various enzymatic properties have been studied. Several differences have been observed in the Km determination, the sensitivity to pH 5 and the activity related to pH between fixed and unfixed enzymes. The role of Pb++ ions in the cytochemical media has also been estimated. It is concluded that several enzymatic differences appear between fixed and unfixed enzymes and that the inhibition by Pb ions is dependent on the buffer and on the amount of substrate used.     

A cerium method for the ultracytochemical localization of monoamine oxidase activity

Summary A cytochemical method based on the complex formation between cerous ions and hydrogen peroxide is described for the ultrastructural localization of monoamine oxidase (MAO). First, the residual MAO activity after fixation was measured by a radiochemical assay technique and was found to be sufficiently retained for cytochemical detection. Although the Tris buffer used in the present method was found to be inhibitory to MAO, considerable activity was still retained after fixation and incubation in Tris.MAO activity, detected as precipitates of cerium perhydroxide, was observed in the mitochondrial outer compartment, mitochondrial cristae and perinuclear space of myocardial cells and endothelial cells of rat heart. MAO activity was also found along the plasma membrane of capillary endothelia. Omission of substrate from the incubation medium or pre-incubation with pargyline, a specific MAO inhibitor, drastically reduced the amount of deposits. The present cerium method seems promising because of its reproducibility and the high electron density of the reaction products.     

Effect of formaldehyde and glutaraldehyde fixation on the immunochemical reactivity of potato virus A with monoclonal antibodies

Potato virus A (PVA) was treated with different glutaraldehyde concentrations at a range of different pH values, and its immunochemical reactivity and integrity was tested in various types of ELISA using a panel of six monoclonal antibodies. Glutaraldehyde fixation was compared with formaldehyde fixation, and there was no significant difference in reactivity between PVA non-treated and treated with formaldehyde, but almost the entire immunochemical activity was lost after treating with glutaraldehyde.     

Effect of fixation on activity and cytochemistry of hydrogenosomal enzymes in Trichomonas vaginalis.

The effect of fixation on the activity of malate dehydrogenase (decarboxylating) and pyruvate synthase was investigated in Trichomonas vaginalis. Subsequently a cytochemical staining method was developed for the demonstration of malate dehydrogenase activity in hydrogenosomes. After fixation of cells in low concentrations of glutaraldehyde and incubation in the presence of malate and the tetrazolium compound 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl)tetrazolium chloride, an electron-dense deposit was produced in the hydrogenosomes. During the whole procedure strictly anaerobic conditions were required. Attempts to develop an analogous procedure for pyruvate synthase failed because even low concentrations of glutaraldehyde strongly inhibited enzyme activity. When cells were fixed in low concentrations of glycolaldehyde and acetaldehyde, a high enzyme activity was retained, but no staining could be achieved. Application of both staining methods to the sapropelic ciliates Trimyema compressum and Plagiopyla nasuta gave negative results.     

Cytochemical localization of ATPase activity in oat roots localizes a plasma membrane-associated soluble phosphatase, not the proton pump

Cytochemical techniques employing lead-precipitation of enzymically released inorganic phosphate have been widely used in attempts to localize the plasma membrane proton pump (H⁺-ATPase) in electron micrographs. Using Avena sativa root tissue we have performed a side-by-side comparison of ATPase activity observed in electron micrographs with that observed in in vitro assays using ATPases found in the soluble and plasma membrane fractions of homogenates. Cytochemical analysis of oat roots, which had been fixed in glutaraldehyde in order to preserve subcellular structures, identifies an ATPase located at or near the plasma membrane. However, the substrate specificity and inhibitor sensitivity of the in situ localized ATPase appear identical to those of an in vitro ATPase activity found in the soluble fraction, and are completely unlike those of the plasma membrane proton pump. Further studies demonstrated that the plasma membrane H⁺-ATPase is particularly sensitive to inactivation by the fixatives glutaraldehyde and formaldehyde and by lead. In contrast, the predominant soluble ATPase activity in oat root homogenates is less sensitive to fixation and is completely insensitive to lead. Based on these results, we propose a set of criteria for evaluating whether a cytochemically localized ATPase activity is, in fact, due to the plasma membrane proton pump.