Zdeněk Lojda

Studies on glycyl-proline naphthylamidase

SummaryGlycyl-proline naphthylamidase (Gly-Pro-Nase) was discovered in 39.4% (±3.4) of peripheral blood lymphocytes of healthy adult humans and in 38.5% (±3.1) of peripheral blood lymphocytes of mini-pigs using glycyl-L-proline-4-methoxy-2-naphthylamide as the substrate and Fast Blue B (diazonium salt to be preferred) or hexazotized New Fuchsin as the coupling agents. The pH optimum of the enzyme is in the alkaline range in the vicinity of neutral. The enzyme activity in lymphocytes is not influenced by 10−3 M EDTA, 10−3 M N-ethyl-maleimide, and 10−3 M MnCl2. It is inhibited to about 50% by 1.4·10−4 M Pb (NO3)2. Individual lymphocytes differ in their activity. In some lymphocytes only one small positive dot in the cytoplsm can be seen. In other cells the number of these dots is greater. In other cases the cytoplasm is overfilled with positively reacting granules and rods. Gly-Pro-Nase in lymphocytes is confined to lysosomes. These organelles may not be its exclusive localization, however.The activity of Gly-Pro-Nase is present in lymphocytes forming E-rosettes with sheep erythrocytes. There is no correlation between the number of bound erythrocytes and the enzyme activity. An unequivocal presence of this enzyme in B-lymphocytes remains to be established. Gly-Pro-Nase is present in differentiated lymphocytes particularly in those bearing ring-shaped nucleoli. It was demonstrated neither in the blastically transformed lymphocytes (after phytohemagglutinine stimulation) nor in epithelial lymphocytes of the human jejunum. Its activity does not go parallel to that of acid esterase or acid phosphatase.Preliminary investigation on peripheral blood lymphocytes of patients sufferring of various diseases of the hemopoetic system revealed a decreased number of positively reacting lymphocytes in chronic lymphatic leukemia (1–20%), normal or slightly elevated values in chronic myeloid leukemia (40–68%), highly elevated values in myelofibrosis (60–78%), and decreased, normal or elevated values in lymphogranuloma (0–70%).Studies on the metabolic as well as diagnostic significance of Gly-Pro-Nase activity in lymphocytes are in progress.

Study on aminopeptidase A

SummaryA comparative biochemical and histochemical investigation of aminopeptidase A (APA, E.C. 3.4.11.7) was carried out. α-Glu-1NA, α-Glu-2NA, α-Glu-MNA and Asp-2NA were used as substrates, Fast Blue B (FBB), hexazotized new fuchsin (HNF) and hexazonium-p-rosaniline (HPR) as coupling agents. Biochemical determination of APA activity was performed in whole homogenates as well as in homogenized freeze-dried cryostat sections of many rat, guinea-pig and human organs. α-Glu-2NA proved to be the best substrate for the biochemical determination of APA activity. It displays favourable kinetic properties and abilities for spectrophotometric as well as fluorometric measurements.Km of rat kidney enzyme amounts to 0.15–0.43 mM, of rat jejunum enzyme 0.35 mM, of myocardial enzyme 0.2 mM and of rat brain enzyme 0.25 mM. The enzyme is activated by Ca2+ and inhibited by EDTA (1 mM) and 1,10-phenanthroline (1 mM). E600, DFP and PCMB (1 mM) did not influence APA activity. The activity was detected in every organ examined. Great organ and species differences were demonstrated. The highest APA activity resides in the kidney and in the small intestinal mucosa.The structural association of APA is not absolutely firm and up to 30% (depending on the organ and eventual pretreatment of sections with chloroform-acetone which impedes the leakage to some extent) escapes into the aqueous incubation solution.The highest values were recorded in phosphate and cacodylate buffers followed by citrate phosphate, citrate, Tris-HCl and acetate buffers. There were no great differences in activities measured at pH 6.5 and 7.2.For the histochemical demonstration of APA activity α-Glu-MNA is the substrate of choice.Km estimated biochemically amounts to 0.3–0.43 mM for the kidney enzyme, 0.35 mM for the rat jejunal enzyme and 0.5 mM for rat brain enzyme.Km estimated by microdensitometric measurements of the reaction product in the brush border of cells of proximal tubules and of jejunal enterocytes at the sites of villi amount to 0.6 and 0.4 mM respectively. The best results were obtained using 2–4 mM concentration of α-Glu-MNA, and satisfactory results using 0.36–0.72 mM concentration. FBB appears to be the best coupling agent in the routine. The most sensitive way to depict APA in sites with low and moderate activity is to use chloroform-acetone pretreated cryostat sections adherent to semipermeable membranes. Sites with high enzyme activity are best revealed upon incubation of chloroform-acetone pretreated cryostat sections adherent to slides. Of the unsubstituted naphthylamine derivative of α-Glu or Asp α-Glu-2NA is superior to α-Glu-1NA or Asp-2NA. In this case HPR or HNF are to be used as coupling agents in cryostat sections adherent to slides.Due to a higher decomposition rate and higher inhibitory influence of diazonium salts on APA activity in the alkaline pH range it is advisable to carry out the reaction at pH 6.5–7.The most important binding sites of APA are:a)Endothelial cells of the capillary bed in all organs (in some organs its venous portion). In the majority of organs this localization represents the unique or at least prevalent binding site of APA. There are differences in the degree of activity in individual organs of the same species and also interspecies differences in the same organ.b)Brush border of small intestinal enterocytes. The activity is present in crypt enterocytes and reaches its maximum in enterocytes of villi. It appears in the duodenum, increases in the aboral direction and after reaching its maximum in the jejunum it declines in the distal ileum. The APA activity is reduced in patients suffering florid coeliac sprue.c)Brush border of cells of proximal tubules in all species.d)Cells of glomeruli (mainly podocytes and mesangial cells) of mouse, rat and human kidney. In the guinea-pig no activity in glomeruli was found.e)Muscle cells of media of arteries of mouse, rat and human coronary arteries, of rat and mouse esophagus, urinary bladder, ductus deferens and uterus.f)Cellular membranes of hepatocytes facing bile capillaries in the mouse and rat liver, sinusoidal lining of guinea-pig liver.g)Intracellularly APA occurs in the Golgi apparatus of jejunal enterocytes and in the cytoplasm of B cells of Langerhans islets in the guinea-pig. The functional significance of APA is discussed in relation to the degradation of angiotensin II (endothelium, kidney) and to other brush border peptidases releasing amino acids which are absorbed (enterocytes). The histochemical demonstration is indispensable for a correct interpretation of biochemical data.

Some remarks concerning the histochemical detection of disaccharidases and glucosidases

SummaryIn the histochemical detection of the disaccharidases and glucosidases the reliability of methods with coupled oxidation of glucose (with various buffers, tetrazolium salts and concentrations of substrates, tetrazolium salts and PMS) and azo-dye methods with the postincubation as well as simultaneous azo-coupling in cryostat sections (unfixed, fixed with Bakers formol and acetone) and frozen sections after fixation in cold Bakerss formol and glutaraldehyde was tested. Various rat organs and human enterobiopsies were used. The methods were modified.Despite the fact that glutaraldehyde and formol fixation does not completely destroy enzyme activities splitting maltose, sucrose, trehalose and lactose (as it could be shown by a simple Glukophan test) the use of the fixed sections is not recommended. Activity of these enzymes is not completely structurally bound and a part of them escapes from the unfixed cryostat sections into the solutions used for rinsing or for incubation. Activities of these enzymes were demonstrated in the content of the rat jejunum as well. The results of the detection of disaccharidases with a coupled oxidation of glucose are dependent on buffer (type and pH), on the tetrazolium salt (type and concentration), on the concentration of phenazine methosulfate and of disaccharides, on the conditions during the incubation (temperature, anaerobic or aerobic conditions, aqueous or gel media) and on the type of sections. With all the substrates used (maltose, sucrose, trehalose and lactose) a positive reaction in the enterocytes (both of rat and human) and in the cells of convoluted tubules in rat kidney was obtained. With lactose the reaction was weak and irregular and could be obtained under anaerobic conditions only. A proximodistal gradient in the rat intestine was revealed. In the detection of lactose the use of galactose oxidase in combination with glucose oxidase decreased the intensity of the staining. In evaluating the validity of the localization the artifacts caused by the diffusion of disaccharidases and by the method with coupled oxidation of glucose were considered, the latter being their main source. By no means such artifacts could be avoided. The positive staining is revealed in the sites of the bound tetrazolium salt where it is contacted by the reduced PMS. No reaction can be obtained in sites lacking affinity for the tetrazolium salts even if they contained an active enzyme. The technique allows at the most the localization on the cellular but not intracellular level. The “disaccharidase granules” of Dahlquist and Brun are artifacts.When the sections are incubated individually with the described gel media or in the incubation chambers the amount of produced formazan may serve as a measure of the activity of the respective disaccharidase. Such technique proved to be of value in investigating the changes of activities of disaccharidases in the jejunum of patients with primary malabsorption syndrome. These activities were reduced in comparison with the normal jejunum.The limitations in localization of the postincubation azo-coupling methods for the deection of glucosidases and galactosidases are much the same as those of the methods with coupled oxidation of glucose. In addition to it the relative substrate specificity of the intestinal disaccharidases has to be considered, because identical enzymes may not be detected with synthetic and natural substrates. Using our new method with hexazo-p-rosaniline in the simultaneous azocoupling an improved localization of 6-Br-2-naphthyl-α-D-glucosidase was achieved. In the enterocytes the enzyme was localized in the microvillous zone and apical part of the cytoplasma.

Indigogenic methods for glycosidases

SummaryAn improved indigogenic method for the detection of β-glucuronidase was devised. The recommended medium consists of 2.4×10−4 M 4-Cl-5Br-3-indolyl-β-D-glucuronide (dissolved in a small amount of dimethylformamide) in 0,1 M acetate buffer pH 5, 3.4×10−3 M potassium ferricyanide and 3.4×10−3 M potassium ferrocyanide.The activity of β-glucuronidase escapes from unfixed or acetone postfixed cold microtome sections into the incubation solution and this leakage cannot be entirely prevented by either the postfixation of sections in cold aldehyde fixatives, or in acetone-formaldehyde-chloralhydrate mixture, or by fixation of freeze-dried sections in isopropylalcohol. No leakage was ascertained from sections prepared from tissue blocks fixed for 24 hours in cold aldehydes. This procedure leads to 70–80% inactivation of enzyme activity in rat spleen, liver and kidney, however. The recommended concentration of ferri-ferrocyanide mixture inhibits the activity by 30% at an average.The overall staining intensity obtained with the improved method in various organs reflects well activities of β-glucuronidase as estimated biochemically in homogenates of these organs. The method enables to demonstrate the lysosomal localization. The distribution pattern obtained with it resembles that obtained with the simultaneous azo-coupling method using naphthol AS-BI-β-glucuronide and hexazonium-p-rosanilin.

Synthetic substrates in the histochemical demonstration of intestinal disaccharidases.

SummaryThe splitting of 6-Br-2-naphthyl-, α-naphthyl-, and 4-Cl-5-Br-3-indolyl-glycosides which proved useful for the assessment of cytological localization of intestinal enzymes in previous studies was investigated using isolated human and rat intestinal disaccharidases as a source of enzyme activities.Previous findings based on histochemical studies were confirmed and extended. 6-Br-2naphthylα-D-glucoside is cleaved by glucoamylase and sucrase-isomaltase. The participatio of trehalase in splitting of this substrate is very low and can be neglected. The mentioned α-glucosidases are responsible for the brush border staining of enterocytes with this substrate when unfixed cold microtome sections are used. Even when a differential heat inactivation of sucrase-isomaltase and of glucoamylase occurs during paraffin embedding (so that the staining in paraffin sections is due mostly to glucoamylase) the use of natural substrates is desirable for a more precise assessment of sucrase-isomaltase activity (but without the possibility of a correct localization).4-Cl-5-Br-3-indolyl-β-D-fucoside is the substrate of choice for the demonstration of lactase. Even when this substrate is split also by “hetero-β-galactosidase” and by acid (lysosomal) β-galactosidase these activities do not disturb the histochemical demonstration of lactase. If however some doubts arise, the inhibition with p-Cl-mercuribenzoate (2 · 10−4 M) is to be emloyed (lactase activity is not inhibited). Due to a low Km and a high Vmax of indolyl-fucoside and due to its extreme stability in solution (which enables to use the substrate solution repeatidly) this substrate is suitable in routine practice even though it is expensive. α-naphthyl- and 4-Cl-5-Br-3-indolyl-β-D-glucosides are split by lactase and β-glucosidase. Due to the fact that the mutual delineation of these activities is not easy and that Km an Vmax for lactase are not so favourable as in the case of fucoside these substrates are not recommended for the assessment of lactase.6-Br-2-naphthyl-β-D-glucoside is the substrate of choice for the histochemical studies concerned with “hetero-β-galactosidase” and 4-Cl-5-Br-3-indolyl-β-D-galactoside for acid β-galactosidase.

The importance of protease histochemistry in pathology

SummaryThe histochemical demonstration of the activities of proteases, based in the majority of cases on the use of discriminating synthetic substrates (aminoacyl or peptidyl derivatives of 4-methoxy-2-naphthylamine [MNA]), has proved very valuable in pathology. Its importance is illustrated by three topics investigated in our laboratory, namely lymphocytes, malabsorption syndrome and atherosclerosis.

Histochemical methods for acid β-galactosidase: Technics for semipermeable membranes

SummaryIndigogenic and azocoupling reactions for the detection of acid β-galctosidase in unfixed cold microtome sections adherent to semipermeable membranes are described. The indigogenic method is the method of choice. The described procedure prevents the leakage of the enzyme activity of sections (the diffusion is limited to the closest surroundings of the actual localization of enzyme activity) and is recommended as a routine method in studies concerning acid β-galactosidase.

β-glucuronidase activity of the aorta in early stages of experimental rabbit atherosclerosis

Summary The effect of daily cholesterol-fat feeding on the β -glucuronidase activity of rabbit aortae was investigated. There was a significant increase in the activity of this enzyme as early as 4 weeks, and also 10 weeks after the beginning of the feeding experiment. An increase in the serum glucosamine level was also observed while the mucoprotein level did not change. It is suggested that these findings reflect early metabolic changes in the aortic connective tissue of cholesterol-fat fed rabbits.

Histochemistry of proteases in ependyma, choroid plexus and leptomeninges

SummaryAminopeptidase M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV) and γ-glutamyl transferase (GGT) were demonstrated histochemically in cryostat sections of the rat brain to show the reaction pattern of ependyma, choroid plexus and leptomeninges. GGT was only demonstrable in the cell membranes of ependymal cells and in the leptomeninges; however, APA, APM and DAP IV showed a variable degree of activity in the capillary endothelium of the choroid plexus as well as in the leptomeninges. On the basis of these results, it is postulated that peptides in the cerebrospinal fluid can be cleaved extraventricularly by the enzymes demonstrated in the leptomeninges.

Remarks on the "OTAN" Reaction

SummaryA series of experiments with the standard OTAN reaction and its modifications was performed in human and animal tissue sections. The coloring obtained in the original OTAN reaction cannot be considered as unequivocal characterisation for hydrophobic and hydrophilic lipids because both kinds of lipids can produce a whole scale of colors ranging from light red to black.The black coloring of hydrophobic lipids can develop after application of α-naphtylamine which does not react only by forming a complex but also in the sense of oxidation-reduction.The red coloring of hydrophobic lipids resistant to prehydrolysis by 1 N NaOH in atheromatous plaques and in arcus senilis corneae could be considered erroneously as presence of the sphingomyelin.Hydrophilic lipids can be stained already after 10 minutes treatment with osmium tetroxide and potassium chlorate mixture. After application of α-naphtylamine a dark brown to brown-black color can develop in some cases which does not signify the presence of hydrophobic lipids. Nonlipid substances can be stained in the OTAN method as well.Modifications of the OTAN method enabling the differentiation of hydrophobic and hydrophilic lipids and lipopigment are described. These consist of pre-extraction with cold acetone or chloroform-methanol or oxidation by periodic acid.