Effects of fixation and substrate protection on the isoenzymes of aspartate aminotransferase studied in a quantitative cytochemical model system

The cytochemical technique of Lee and Torack for the demonstration of aspartate aminotransferase activity was tested on a model system consisting of either total liver homogenate or the mitochondrial or soluble cytoplasmic fraction, incorporated in polyacrylamide film. After incubation of portions of film in a medium of α-ketoglutarate, L-aspartate, and lead nitrate, the lead oxaloacetate formed was converted to lead sulfide. The absorbance determined at 520 nm in a film spectrophotometer and expressed in terms of unit weight of film provided a measure of the contained enzymatic activity, and was directly proportional to the concentration of chemically determined oxaloacetate in the film. Both mitochondrial and "soluble" isozymes of aspartate aminotransferase reacted with the cytochemical media to a quantitatively similar degree, but were considerably inactivated after 15 min of treatment with 1% glutaraldehyde or 3.7% formaldehyde in imidazole buffer, the rate of inactivation being greater for the soluble isozyme. Application of the principle of substrate protection delayed inactivation. Thus, for both isozymes the rate of inactivation decreased if ketoglutarate was added to the fixative. Similarly, it was shown that the optimal incubation medium for the demonstration of the soluble isozyme must contain 4 mM of α-ketoglutarate and 20 mM of L-aspartate. Under these conditions the turnover-number for the cytochemical system is 70% of the value obtained from biochemical estimations. Cytochemical K_m values differed for each isozyme and were in accord with values determined by biochemical techniques, indicating that the model system can be used as a link between biochemical and cytochemical data in enzymatic studies.     

A comparison of directed evolution approaches using the beta-glucuronidase model system

Protein engineers can alter the properties of enzymes by directing their evolution in vitro. Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science. We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity. Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen. GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations. The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen. Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele. These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.     

Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974). Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.     

Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

Summary A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974).Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.This word was performed while one of us (Dr. Andrä) was in receipt of a visitor grant from the Netherlands Organization for the Advancement of Pure Research (ZWO)     

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach

Summary A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substrances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.     

Chromatin organization in frog Purkinje neurons during the annual cycle: cytochemical and ultrastructural studies

Feulgen-DNA microdensitometric and propidium Iodide-DNA (PI-DNA) microfluorometric evaluations were made in Purkinje cells of cerebella from hibernating frogs and from active frogs. Higher Feulgen-DNA/distribution area ratios and lower PI-DNA values were found in the hibernating frog (both at high and low fluorochrome concentrations). These data indicate a higher degree of chromatin compactness. During the activity period, the Feulgen-DNA/distribution area ratios are lower and the Feulgen-DNA content is higher; the corresponding higher PI-DNA values indicate a lower degree of chromatin condensation. Histochemical and ultrastructural data show clearly different distribution patterns of chromatin and ribosomes in the two periods. The overall results underscore lower activity of neurons and a greater homogeneity within the Purkinje cell population during hibernation.     

The cytochemical demonstration of beta-glucuronidase in colon neoplasms of rats exposed to azoxymethane

Colon tumors induced with azoxymethane in male Fischer rats were cytochemically analyzed for beta-glucuronidase using naphthol AS-B1 glucuronide (6-bromo-2-hydroxy-3-naphthoyl-O-anisidine) as a substrate and hexazonium pararosanin as a diazo reagent. This method effectively localizes the bulk of beta-glucuronidase in the surface epithelium, the lamina propria and in the endothelial cells of the lymphoid sinuses and postcapillary venules. Polypoid lesions, adenocarcinomas and mucinous adenocarcinomas show no difference in the amount or in the localization of beta-glucuronidase; however, mucinous adenocarcinomas show a slight increase in the amount of beta-glucuronidase. The few tumors that did metastasize to lymph nodes did not show any difference in their enzyme patterns. Intestinal crypts that show a change in size and shape have a definite increase in beta-glucuronidase activity. An increase in the activity of this enzyme can also be seen in well defined neoplasms as opposed to normal areas of the colon.     

Further cytochemical studies on the perichromatin granules

Summary The perichromatin granules were studied in hepatocytes of experimental rats injected with cycloheximide because the increased number of these nuclear components after such treatment facilitated their cytochemical investigation. Most perichromatin granules were sensitive to the digestion with pepsin and ribonuclease. In contrast, small population of perichromatin granules was resistent to such digestion under conditions which remove known RNA containing components such as ribosomes, nucleolar RNP components and interchromatin granules. The size of these resistent perichromatin granules was reduced and they consisted of filaments the width of which was similar to that of filaments in the chromatin. Moreover, a small population of perichromatin granules was sensitive to the digestion with pepsin and deoxyribonuclease. The size of these granules was only slightly reduced. All these observations indicate that most perichromatin granules contain the RNA and some the DNA. A possibility also exists that the perichromatin granules might contain both RNA and DNA but in various proportions. In addition, partial digestion with pepsin followed by a complete digestion with ribonuclease and deoxyribonuclease removed perichromatin granules as well as other nucleoprotein structures. On the other hand, such digestion facilitated the visualization of the nuclear and cytoplasmic skeleton (matrix) in situ.Dedicated to the memmory of Dr. W. Bernhard     

Selection of an animal model for implant fixation studies: anatomical aspects.

A number of different animal models have been employed by investigators to study the biology of the bone-cement interface as it relates to the problem of hip implant loosening in humans. This study compares to the human three species (baboon, dog, and sheep) currently under use as experimental animal models from an anatomical point of view. A number of parameters, important for the dimensional design of a femoral prosthesis, loads at the hip joint and its subsequent performance, were used for comparing external and internal femoral anatomy. The baboon and dog femora were found to be most similar to the human femur in their external anatomy. The quantification of cancellous bone distribution within the medullary canal revealed that, of the species studied, the sheep femur provided the least support to the prosthesis. The results suggest that the dog and baboon are anatomically appropriate for studying hip implant biomechanics experimentally. Thus, from an anatomical point of view, the current extensive use of the dog as an experimental animal appears appropriate.     

Immunohistochemistry of enkephalins: model studies on hapten-carrier conjugates and fixation methods.

For immunohistochemical demonstration of the enkephalin octapeptide Met5-enkephalin-Arg6-Gly7-Leu8, the peptide was conjugated with a carrier protein using either glutaraldehyde or 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide as coupling agent. Antisera were raised in rabbits and their specificity was studied using the immunoblotting technique. The results suggest that glutaraldehyde selectively couples the amino terminus of the peptide to the carrier protein, while carbodiimide coupling produces a mixture of specificities. Accordingly, antiserum raised against the glutaraldehyde-induced conjugate specifically recognized the peptide carboxyl terminus and allowed immunohistochemical distinction of the octapeptide from other closely related opioid peptides, such as Leu5- and Met5-enkephalin, Met5-enkephalin-Arg6-Phe7, and Phe1-Met2-Arg3-Phe4-NH2. In contrast, antiserum raised against the carbodiimide-induced octapeptide conjugate showed a mixture of specificities. Addition of glutaraldehyde to the fixative enhanced octapeptide immunoreactivity in several tissues and revealed a previously unknown nerve system in the pituitary gland. These results support the idea that optimal immunohistochemical demonstration of small molecules, which requires conjugation to a carrier protein, is obtained when the coupling agent is included in the fixative so as to induce the actual coupling reaction during fixation.     

Substrate protection of immobilized glucose isomerase

Using commercial immobilized glucose isomerase (SWETASE(R), Nagase Co.), the effect of substrate protection on enzyme deactivation has been studied in a batch manner. The data analysis was carried out based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrates was also considered. The protection factor was proposed to elucidate the dependence of the degree of substrate protection. The existence of the protection of glucose isomerase by the substrates has been verified experimentally. Also, the enzyme-substrate complex deactivates with a decay constant which is one-half that of the free enzyme. Theoretical analysis of enzyme deactivation with substrate protection offers an effective understanding which is essential for enzyme replacement and process optimization.