Practical utility of an antibody specific to a synthetic peptide corresponding to the N-terminal amino acid sequence of human urine DNAse I for genetic analysis of human DNase I isozymes

An antibody specific to a synthetic peptide corresponding to the N-terminal 27 amino acid residues of human urine DNase I (anti-DNase I peptide) was obtained. The antibody did not inhibit the activity of the enzyme, but reacted well with the enzyme upon immunoblotting following electrophoresis. The urine DNase I isozyme patterns detected using this antibody were almost identical to those produced with an antibody specific to purified DNase I. Therefore, the anti-DNase I peptide antibody should prove to be valuable for genetic analysis of human DNase I isozymes.     

Human urine deoxyribonuclease II (DNase II) isoenzymes: a novel immunoaffinity purification, biochemical multiplicity, genetic heterogeneity and broad distribution among tissues and body fluids.

Deoxyribonuclease II (DNase II) was purified from the urine of a 48-year-old male (a single individual) using a column chromatography series, including concanavalin A-agarose and an immunoaffinity column utilizing anti-human spleen DNase II antibody, and was then characterized. Based on the catalytic properties of the purified enzyme, we have devised a technique of isoelectric focusing by thin-layer polyacrylamide gel electrophoresis (IEF-PAGE) combined with a specific zymogram method, for investigating the possible molecular heterogeneity of human DNase II. DNase II in urine as well as the purified form was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 5-7. Since sialidase treatment of the urine sample induced simplification of the isoenzyme patterns with diminishment of anodal bands, it was clear that the multiplicity of the enzyme was in part due to differences in the sialic acid content. On screening of DNase II isoenzyme patterns in urine samples from more than 200 Japanese individuals, only the common isoenzyme pattern was observed and no electrophoretic variations were detected. However, genetic studies of urinary enzyme activity and comparative studies on the activity in urine, semen and leukocytes from the same individuals suggest that the enzyme activity level of DNase II may be under genetic control. The enzyme was widely distributed in human tissues and showed high activities in secretory body fluids such as breast milk, saliva, semen and urine, and leukocyte lysates.     

Purification and characterization of genetically polymorphic deoxyribonuclease I from human kidney.

Deoxyribonuclease I (DNase I) was purified about 850,000-fold from human kidney using a rabbit anti-human urine DNase I antibody and sensitive DNase I activity assay. On SDS-PAGE, the purified kidney DNase I gave a single major band, and its molecular mass was estimated to be 38,000 Da. The activity of purified kidney DNase I was dependent on the presence of Mg2+ and Ca2+. G-Actin inhibited the activity, as did the anti-urine DNase I antibody. The properties of the kidney DNase I were the same as those of urine DNase I.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

Novel detection method for human protein C inhibitor (PCI) using immunoblotting with antibodies specific to synthetic peptides corresponding to the N-terminal and C-terminal amino acid sequences of human PCI.

Three types of antibody, which were specific to the purified human protein C inhibitor (PCI) and to two synthetic peptides, which corresponded to the N-terminal 15 amino acid residues (PCI-N) and C-terminal 15 residues (PCI-C) of PCI, were produced. Plasma PCI patterns, which were shown by means of polyacrylamide gel isoelectric focusing followed by immunoblotting with the antibody to purified PCI, were almost identical to those obtained with the antibodies to the peptides, PCI-N and PCI-C. The latter antibodies against the synthetic peptides proved very useful for biochemical or genetic analysis of human plasma PCI, as such peptide antigens could be produced without any of the tedious procedures necessary to obtain purified PCI as an immunogen.     

Carp hepatopancreatic DNase I: biochemical,molecular, and immunological properties

A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.     

Genetic polymorphism of human serum ribonuclease I (RNase I).

One of the human urinary ribonucleases (RNases) was isolated and purified to homogeneity (SDS-PAGE) by means of a series of column chromatographies. The enzyme, designated RNase 1, is a glycoprotein with a molecular weight of approximately 16,000. Rabbit antibody to the purified RNase 1 reacted with human urine and sera, as well as with the purified RNase 1. The genetic polymorphism of serum RNase 1 was studied by polyacrylamide gel isoelectric focusing (IEF-PAGE) in a pH range of 5-8, followed by immunoblotting with antisera specific for RNase 1. Two common phenotypes, RNASE1 1 and RNASE1 1-2, were easily recognized. The homogeneous phenotype, RNASE1 1, consisted of four major bands with different pI values, and the heterogeneous phenotype, RNASE1 1-2, was presumed to represent a mixture of each of the homogeneous phenotypes 1 and 2; however, the other homogeneous phenotype, RNASE1 2, was not detected in our samples. Family studies are in agreement with an autosomal codominant transmission of the two alleles. Population studies indicate that the frequencies of the RNASE 1 and RNASE1 2 alleles are .988 and .012, respectively.     

The zymogram method for detection of ribonucleases after isoelectric focusing: analysis of multiple forms of human, bovine, and microbial enzymes.

A zymogram method for detection of in situ ribonuclease (RNase) activity, combined with isoelectric focusing in a thin layer of polyacrylamide gel (IEF-PAGE), has been developed. After incubation with a dried agarose film containing substrate RNA, ethidium bromide, and an appropriate reaction buffer, which was placed tightly on the top of the focused gel, sharp and distinct dark bands corresponding to RNase isoenzymes on a fluorescent background appeared under uv light. Addition of urea to the IEF-PAGE gel at a final concentration of 4.8 M permitted optimal focusing of the RNases. This method had not only a high sensitivity of less than 0.1 ng purified RNase A, but also a high band resolution compared with the immunostaining method. It was also useful for analysis of purified enzymes, including bovine pancreatic RNases and two types of human urine RNase as mammalian enzymes, and RNases T1 and T2 as microbial enzymes, as well as for detection of RNases present in crude tissue extracts, resulting in more detailed elucidation of the multiplicity of these enzymes.     

Biochemical and genetic studies on GP43, a 43-kD glycoprotein detected immunologically in human urine and serum

A new and previously undescribed glycoprotein with a molecular weight of 43,000 has been isolated from human urine. This protein, designated GP43; copurified with ribonuclease, which has the same molecular weight, but ribonuclease activity was removed by passage through an affinity column of agarose-5'-(4-aminophenyl phosphoryl) uridine 2'(3') phosphate. GP43 contains about 5.9% neutral sugar, 2.3% hexosamine, and 1.6% sialic acid. A rabbit antibody to the purified GP43 reacted with human urine and serum as well as with the purified GP43. The genetic polymorphism of GP43 was then studied in desialylated human serum samples by urea-polyacrylamide gel isoelectric focusing, followed by immunoblotting with the specific antibody for GP43. Three common phenotypes, designated GP43 1, 1-2, and 2, were easily recognized using this technique and represented homozygosity or heterozygosity for two autosomal codominant alleles, GP43*1 and GP43/2. The frequencies of the GP43*1 and GP43*2 alleles in a Japanese population were 0.7683 and 0.2317, respectively.