One-step purification of mammalian deoxyribonucleases I and differences among pancreas, parotid, and pancreas-parotid (mixed) types based on species- and organ-specific N-linked glycosylation

Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described. This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of the enzyme.     

One-step purification of mammalian deoxyribonucleases I and differences among pancreas,parotid, and pancreas-parotid (mixed) types based on species-and organ-specific N-linked glycosylation

Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described. This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of the enzyme.     

Isolation, characterisation and crystallization of deoxyribonuclease I from bovine and rat parotid gland and its interaction with rabbit skeletal muscle actin

A purification procedure is described yielding DNase I from bovine and rat parotid glands of high homogeneity. The apparent molecular masses of the DNases I isolated have been found by sodium dodecyl sulfate/polyacrylamide gel electrophoresis to be 34 and 32 kDa for bovine and rat parotid DNase I, respectively, and thus differ from the enzyme isolated from bovine pancreas (31 kDa). By a number of different criteria concerning their enzymic behaviour, the isolated enzymes could be clearly classified as DNases I, i.e. endonucleolytic activity preferentially on native double-stranded DNA yielding 5'-oligonucleotides, a pH optimum at about 8.0, the dependence of their enzymic activity on divalent metal ions, their inhibition by 2-nitro-5-thiocyanobenzoic acid and by skeletal muscle actin. Comparison of their primary structure by analysis of their amino acid composition and also two-dimensional fingerprints and isoelectric focusing indicate gross similarities between the enzymes isolated from bovine pancreas and parotid, but distinct species differences, i.e. between the enzymes isolated from bovine and rat parotid. All the DNases I are glycoproteins. From bovine parotid DNase I crystals suitable for X-ray structure analysis could be obtained. The DNases I from both parotid sources specifically interact with monomeric actin forming 1:1 stoichiometric complexes. Their binding constants to monomeric actin differ, being 2 X 10(8) M-1 and 5.5 X 10(6) M-1 for bovine and rat parotid DNase I, respectively. Only the enzyme isolated from bovine sources is able to depolymerize filamentous actin.     

Deoxyribonuclease I in mammalian tissues. Specificity of inhibition by actin

Enzymes of the DNase I class, similar to bovine pancreatic DNase I with respect to molecular weight and ionic and pH requirements, were found in various tissues of the rat. Their analysis was facilitated by a method for detection of nucleases in crude extracts after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and subsequent renaturation of the enzymes. High levels of DNase I were found in digestive tissues, such as the parotid and submaxillary salivary glands and the lining of the small intestine., Appreciable levels were present in the lymph node, kidney, heart, prostate gland, and seminal vesicle. No activity was found in pancreatic extracts. However, under some conditions, tissues rich in proteases gave poor recovery of DNase I. Fourteen other tissues showed little or no DNase I. Inhibition of various DNase I enzymes by rabbit muscle actin was examined both in gels and in solution. Actin inhibited the bovine parotid DNase I as well as the bovine pancreatic enzyme, but actin did not inhibit any of the DNase I enzymes of the rat. This species specificity of actin inhibition makes it unlikely that the very strong association between monomeric actin and bovine DNase I is of general significance for cellular function.     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

Multiple forms of deoxyribonuclease I

Summary This article will review recent progress on the purification of DNase I (E.C.3.1.4.5) from various sources and the characterization of multiple forms of the enzyme. The chemical basis of the multiple forms in bovine pancreas will be discussed in detail, while for other DNases, including those in ovine pancreas, bovine, mouse and rat parotid, and malt, only the evidence for multiplicity will be presented.     

Notes on Technic: Differential staining of Fungus and host cells Using a modifciation of Pianeze IIIB

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Staining Secretory Capillaries of Exocrine Glands with Techniques for Specific Phosphatases

Intercellular secretory capillaries in parotid glands, eccrine sweat glands and intracellular secretory capillaries in parietal cells of gastric glands were demonstrated histo-chemically by the use of the Wachstein-Meisel adenosinetriphosphatase (ATPase) technique in the rabbit, rat and guinea pig. However, with the Wachstein-Meisel 5-nucleotidase technique, secretory capillaries were not stained. For parotid glands, optimal incubation in ATPase substrate mixture was: in rabbit, 15 min; in rat, 2.5 hr; and in guinea pig, 2 hr. For eccrine sweat glands, optimal incubation was 15 min in rabbit, 30 min in rat and 15 min in guinea pig. For parietal cells of gastric glands, optimal incubation was 3 hr for all three species. Secretory capillaries were best demonstrated in the parotid by using rabbit tissue; in eccrine sweat glands, with rat tissue, and in parietal cells, guinea pig tissue. Since ATPase activity in cell membranes of secretory cells may play a part in the mechanism of transport of secretory products from their place of formation in the acini to the excretory ducts, the Wachstein-Meisel ATPase technique can therefore be used successfully for staining secretory capillaries in many of the exocrine glands of laboratory mammals.     

Molecular, biochemical and immunological analyses of porcine pancreatic DNase I.

Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.     

The immunological and structural comparisons of deoxyribonucleases I. Glycosylation differences between bovine pancreatic and parotid deoxyribonucleases

A rabbit antiserum against bovine pancreatic DNase A is used to study the immunological reaction of DNases I. As shown by double immunodiffusion, bovine pancreatic DNases A, B, C, and D are immunologically identical, so are DNases from bovine pancreas and parotid and from ovine pancreas. These DNases also behave similarly in immunotitration of DNase activity and all are tightly bound to the immunoaffinity medium, requiring an acidic buffer with 10% ammonium sulfate to dissociate. On the other hand, porcine pancreatic and malted barley DNases that do not form precipitin lines remain active in solution with the antibody; however, in spite of the lack of inhibition these DNases are retarded (but not tightly bound) in immunoaffinity chromatography, suggesting interaction with the antibody. In thin layer isoelectric focusing, the parotid DNase, purified with the immunoaffinity technique, shows only two major active components whose isoelectric points correspond to those of DNases A and C of bovine pancreas. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of parotid DNase is 34,000, approximately 3,000 more than that of the pancreatic enzyme. However, both parotid and pancreatic DNases have the same NH2-terminal leucine, an identical COOH-terminal amino acid sequence, nearly identical amino acid compositions, and almost the same peptide maps. The molecular weight difference is due to differences in the carbohydrate side chains. Results of peptide analyses indicate that parotid DNase contains two glycopeptides; pancreatic DNase has only one. In addition, both parotid glycopeptides contain glucosamine and galactosamine while the pancreatic glycopeptide has only glucosamine.     

The inhibition of bovine and rat parotid deoxyribonuclease I by skeletal muscle actin. A biochemical and immunocytochemical study.

Rat and bovine parotid gland and pancreas contain deoxyribonuclease I (DNAase I) activities in different amounts. The DNAase I activity in tissue homogenates of bovine and rat parotid gland can be inhibited by addition of monomeric actin, as with the enzyme of bovine pancreas. The isolated DNAase I species from bovine and rat parotid gland differ in their molecular weights and also in their affinities for monomeric actin, being lowest for rat parotid DNAase I (5 X 10(6)M(-1). Antibodies raised against rat and bovine parotid and bovine pancreatic DNAase I can be used to study the subcellular localization of DNAase I in these tissues by indirect immunofluorescence. DNAase I was found to be confined solely to the secretory granules of the tissue from which it was isolated.     

Tissue distribution and characterization of peptide C-terminal alpha-amidating activity in rat

The C-terminal alpha-amide formation of peptides is one of the most important events in prohormone processing. Recently, we developed a simple and sensitive assay for detecting alpha-amidating activity in tissues by using (125I)-Ac-Tyr-Phe-Gly as a substrate. Using this assay method, we have determined the tissue distribution of alpha-amidating enzyme activity in adult male rat. High concentrations of alpha-amidating activity were found in pituitary, brain, thyroid, gastrointestinal tract, pancreas, heart, submaxillary glands and parotid glands. Alpha-amidating enzyme activities in all tissues examined exhibit very similar copper and ascorbate requirements, pH dependence, and behavior on gel-filtration.     

Distribution and properties of arginase in the salivary glands of four species of laboratory mammals

Important progress in arginine metabolism includes the discovery of widespread expression of two isoforms of arginase, arginase I and II, not only in hepatic cells but also in non-hepatic cells, and the formation of nitric oxide, a widely distributed signal-transducing molecule, from arginine by nitric oxide synthase. Possible physiological roles of arginase may therefore include regulation of nitric oxide synthesis through arginine availability for nitric oxide synthase. In this paper, arginase was investigated in the submandibular, sublingual, and parotid glands of rat, mouse, guinea pig, and rabbit. From their arginase contents, the salivary glands of these species were divided into two groups. Variable levels of arginase activity were detected in the salivary glands of mouse and rat. However, salivary glands of rabbit and guinea pig had almost no arginase activity. The presence of nitric oxide synthase has been reported in all the salivary glands used in this study. Therefore, one of the important findings was the presence of species specificity in the co-localization of arginase and nitric oxide synthase in the salivary glands of the four species. The highest specific activity of arginase was found in mouse parotid gland. In rat, considerable arginase activity was detected in all three glands, at 3.6–7.3% of that in rat liver. In rat submandibular gland, arginase was detected in both cytosolic and particulate fractions. In addition, arginase was detected in isolated acinar cells, but not in duct cells. Experiments on the intracellular distribution and the effects of the arginase inhibitors ornithine and N-hydroxy-L-arginine (NOHA), suggested the presence of both arginase I and arginase II in rat submandibular gland.Abbreviations cGMP cyclic guanosine 3,5-monophosphate - NO nitric oxide - NOHA N-hydroxy-L-arginine - NOS nitric oxide synthase Communicated by I.D. Hume     

Inter- and intraspecies comparisons of fibre type distribution and of succinate dehydrogenase activity in type I, IIA and IIB fibres of mammalian diaphragms

Fibre types in the costal region of the diaphragm muscle of several mammalian species with widely different respiratory rates were examined microphotometrically for succinate dehydrogenase (SDH) activity. Mean activities indicated no significant (p greater than 0.05) difference between the type I and IIA fibres for any of the species examined. SDH activities in type IIB fibres were significantly lower (p less than 0.05) than either the type I or type IIA fibres in the cat, guinea pig, rat and rabbit whereas in the mouse no difference was found. The dog had no classical type 1B fibres. Analysis of the distribution of SDH activities by fibre type indicated a wide scattering of scores with no distinct separation between fibre types. Large differences in SDH activity were noted between species. Mean SDH activities were highest in the mouse and rat, intermediate in the rabbit and guinea pig and lowest in the cat and dog. These data suggest an association between respiratory rate and aerobic oxidative potential of the various fibre types in diaphragms of the species examined.     

Proteolytic activity of the enzyme complex of the mammalian pancreas in comparison with pancreatin

The proteolytic activity and thermal stability of the enzyme complex of cell suspension from pig and bovine pancreas glands was compared with those of pancreatin. The enzyme complex displayed the highest thermal stability and activity at 50 degrees C. The kinetic constants, energies of activation and inactivation of the enzyme complex, and pH optimum (7.0 +/- 0.1) at which this complex had the maximum proteolytic activity were determined. Pancreatin had a pH optimum of 8.0 +/- 0.1.     

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.     

Inter- and intraspecies comparisons of fibre type distribution and of succinate dehydrogenase activity in type I,IIA and IIB fibres of mammalian diaphragms

Summary Fibre types in the costal region of the diaphragm muscle of several mammalian species with widely different respiratory rates were examined microphotometrically for succinate dehydrogenase (SDH) activity. Mean activities indicated no significant (p>0.05) difference between the type I and IIA fibres for any of the species examined. SDH activities in type IIB fibres were significantly lower (p<0.05) than either the type I or type IIA fibres in the cat, guinea pig, rat and rabbit whereas in the mouse no difference was found. The dog had no classical type 1B fibres. Analysis of the distribution of SDH activities by fibre type indicated a wide scattering of scores with no distinct separation between fibre types. Large differences in SDH activity were noted between species. Mean SDH activities were highest in the mouse and rat, intermediate in the rabbit and guinea pig and lowest in the cat and dog. These data suggest an association between respiratory rate and aerobic oxidative potential of the various fibre types in diaphragms of the species examined.     

Characteristics of sialidase in the rat salivary glands

Using 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as substrate, we measured sialidase activity in the salivary glands and other organs of the rat. The pH optima of salivary gland sialidase were between 4.0 and 4.5, which were similar to those of the enzyme in the brain, liver and kidney. Among the salivary glands, the submandibular one showed the highest sialidase activity followed by the parotid and the sublingual glands. However, sialidase activity in these glands was lower when compared with the activity in the brain, liver and kidney. From the subcellular distribution study, salivary gland sialidase was found to be mainly localized in the lysosomes. The pH optima of the lysosomal sialidase of the salivary glands were between 4.0 and 4.5; and Km values for 4MU-NeuAc approximately 0.09 mmol/l. In the submandibular and parotid glands, a soluble sialidase with a different pH optimum (5.5) and Km value (0.25 mmol/l) was also detected.     

Comparison of proteolytic activities of the enzyme complex from mammalian pancreas and pancreatin

The proteolytic activity and thermal stability of the enzyme complex of a cell suspension from pig and bovine pancreas glands was compared with those of pancreatin. The enzyme complex displayed the highest thermal stability and activity at 50°C. The kinetic constants, energies of activation and inactivation of the enzyme complex, and pH optimum (7.0 ± 0.1) at which this complex had the maximum proteolytic activity were determined. Pancreatin had a pH optimum of 8.0 ±0.1.