IgA1 is the premier serum glycoprotein recognized by human galectin-1 since T antigen (Galbeta1-->3GalNAc-) is far superior to non-repeating N-acetyl lactosamine as ligand

Human heart galectin-1 (HHL) was separated by high pressure liquid chromatography from endogenous glycoproteins co-purified with it during affinity chromatography. These glycoproteins offered excellent ligands for HHL binding and were rich in T antigen (Galβ1 → 3 GalNAc-) of O-linked oligosaccharides. In enzyme linked lectin assay and hemagglutination inhibition assay, human IgA1, bovine fetuin and other O-glycosylated T antigen-bearing glycoproteins bound to the lectin efficiently in contrast to single N-acetyl lactosamine (LacNAc)-bearing N-linked oligosaccharides released from them and to IgG which is not O-glycosylated. HHL binding to IgA1 and fetuin was unaffected by removal of their N-linked oligosaccharides by -mannosidase. When immobilized, O-glycosylated serum proteins but not IgG could capture HHL from its solutions. Desialylated or polymeric IgA1 was better inhibitor than monomeric IgA1. The findings suggest a possible role for galectin-1 in anchoring of microbial and cancer cells known to be rich in T antigen, in high serum IgA1 turn over and in tissue sequestering of IgA1 immune complexes especially after their microbial desialylation in IgA nephropathy and other immune complex-mediated disorders.     

Determination of angiotensin converting enzyme inhibitory activity by high-performance liquid chromatography/electrospray-mass spectrometry

A sensitive and rapid method for determination of angiotensin converting enzyme (ACE) inhibitory activity was developed based on a combination of enzymatic reaction followed by high performance liquid chromatography/electrospray-mass spectrometry (HPLC-ESI-MS) determination of its product. The most commonly used substrate hippuryl-histidyl-leucine (HHL) or hippuryl-glycyl-glycine (HGG) hydrolysis catalyzed by purified rabbit lung ACE or human plasma ACE was investigated in the presence of benazeprilat. The incubation time was 8 min for purified lung ACE, and 16 min for human plasma ACE. The produced hippuric acid (HA) was separated from substrate HHL or HGG by HPLC on a C(18) column with isocratic elution within 6.5 min, and quantified by electrospray ionization mass spectrometry (ESI-MS) with p-phthalic acid as an internal standard (IS). The limit of detection of HA was 6.0 ng/ml. HHL or HGG hydrolysis catalyzed by purified lung ACE displayed excellent accuracy and reproducibility. The small total reaction volume, the low concentration of substrate, and the simple treating procedures present the advantages of the new method. Furthermore, the total time of the whole procedure for one sample with the novel method is less than 1/2 of that of the conventional HPLC or spectrophotometry method, while the accuracy and the precision of the new method are almost the same as the conventional HPLC method with UV detection.     

Distribution of two urinary ribonuclease-like enzymes in human organs and body fluids

In order to determine the distribution of two human urinary RNase (RNase Us and RNase UL)-like enzymes in human tissues and body fluids, enzyme immunoassay systems were established using rabbit anti-RNase sera. The sensitivity of the assay systems was of similar order to that of radioimmunoassay systems previously reported. In the enzyme immunoassay, the cross reactivities of anti-RNase UL serum towards RNase Us, bovine kidney RNase K2, bovine RNase A, and bovine seminal RNase Vs were less than 1%. The cross reactivity of anti-RNase Us-serum towards RNase UL was less than 0.5% and cross reactivities were minimal for RNase A, RNase K2, and RNase Vs. The RNase levels in human organs and body fluids were measured by enzyme immunoassay. In milk, semen and saliva, only RNase UL-like enzyme was found. Both RNase Us- and RNase UL-like enzymes were found in kidney, stomach, and pancreas and the RNase Us/RNase UL ratios were 0.49, 1.35, and 0.34, respectively. In lung, liver, spleen, and leukocytes, most of the RNase activity was accounted for by RNase Us-like enzyme. The activity of RNase Us-like enzyme was especially high in lung, spleen, and leukocytes. The crude extracts of several tissues and body fluids were separated by phosphocellulose column chromatography and the contents of the two urinary RNase-like enzymes were determined by enzyme immunoassay. In stomach, kidney, pancreas, and serum, both enzymes were present in multiple forms. In spleen and lung, both the major RNase (RNase Us) and minor RNase (RNase UL) existed in two forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid and simple spectrophotometric assay of angiotensin-converting enzyme.

A rapid, simple, and accurate method for the chemical assay of anglotensin-converting enzyme has been developed. The method relies on previously published method for spectrophotometric assay of angiotensin-converting enzyme activity and on the use of 2,4,6-trichloro-s-triazine (TT) as a colorimetric reagent of hippuric acid (N-benzoylglycine). When 3% TT in dioxane was added to the incubation medium of the angiotensin-converting enzyme after stopping the incubation by the immersion of the test tubes in a boiling-water bath, the absorbance at 382 nm increased linearly as a function of both enzyme concentration and incubation time. Neither hippuryl-l-histidyl-l-leucine (HHL, substrate for this assay system) nor histidyl-leucine was positive in color reaction with TT. Accordingly, this method does not require any procedures for separation of hippuric acid from HHL. The enzyme activity was found to be highest at pH 8.3, at chloride ion concentration of 600 mm, and at HHL concentration of 3 mm, when the 5000g supernatant fluid of the rat lung was used.     

Phylogenetic conservation of epitopes in mammalian aldose reductase: application to immunoquantitation

Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific by Western blot analysis of kidney homogenates. In addition, the antiserum (alpha-BKALR2) reacts with a single electrophoretic species in homogenates from rabbit, porcine, and human kidney. ALR2 has been detected in homogenates of bovine kidney, heart, brain and lens, and estimation of the enzyme level in these tissues was accomplished by densitometric analysis of Western blots. Standard curves using highly purified bovine kidney ALR2 were linear in the range of 5-100 ng; a similar sensitivity was seen in tissue homogenates. The results presented here for the ALR2 level in bovine tissues (kidney greater than heart greater than brain greater than lens) are in agreement with literature values for those tissues from which the enzyme has previously been purified. The interspecies similarity in electrophoretic mobility and the retention of antibody reactivity suggest extensive phylogenetic epitope conservation in mammalian aldose reductase.     

Immunoquantitation of aldose reductase in human tissues

Rabbit antibodies raised against bovine kidney aldose reductase (ALR2) were shown to be monospecific for human ALR2 by Western blot analysis of human muscle homogenates. The human enzyme was detected, by reaction with the antiserum (alpha-BKALR2), in homogenates of adrenal gland, muscle, lens, brain, testes, kidney, and placenta, but not in erythrocytes or leukocytes. The amount of enzyme in each tissue was determined by densitometric analysis of autoradiographs of Western blots probed with alpha-BKALR2 and [125I]protein A. Standard curves of radiographic intensity versus amount of purified human muscle ALR2 were linear in the 20 to 200-ng range; a similar sensitivity was seen in tissue homogenates containing up to 675 micrograms total protein. The results presented here for the ALR2 level in human tissues (adrenal greater than muscle greater than lens approximately brain approximately testes greater than kidney approximately placenta) are in agreement with literature values for those tissues from which the enzyme has previously been purified. A notable exception was the absence of detectable ALR2 in human erythrocytes. A quantitative comparison of immunoradiographic response showed that bovine kidney ALR2 was about sevenfold more reactive with a alpha-BKALR2 compared to the human muscle enzyme.     

Physicochemical characteristics of homogeneous bovine lung angiotensin I-converting enzyme. Comparison with human serum enzyme

Angiotensin I-converting enzyme was purified to electrophoretic homogeneity (12 units/mg) from bovine lung tissue and from human serum using an affinity gel described previously (Harris et al., (1981) Anal. Biochem. 111, 227-234). The isoelectric point (4.5), molecular weight (145 000), S20,W (8.1), amino acid composition and carbohydrate content of the lung enzyme are all similar to the values obtained for the human serum enzyme. The NH2-terminus of the lung enzyme (Ala) is different from that of the serum enzyme (Tyr) but the COOH-terminal sequences are identical (-Leu-Ser-OH). Pure bovine lung enzyme was reduced and carboxyamidomethylated with iodo (14C1) acetamide to the extent predicted by the number of cysteine residues. Since no radioactivity was incorporated into denatured enzyme that was not reduced, all of the cysteine residues must be in the form of disulfide bonds. Reverse-phase HPLC was used to separate peptides obtained from the lung enzyme after degradation with either trypsin or cyanogen bromide. The number of peptides resolved (42 after trypsin, 31 after cyanogen bromide), were only 20% fewer than the number predicted from the amino acid analysis and therefore the possibility that the converting enzyme (a single polypeptide chain) might be a fused dimer is excluded.     

Studies on cytosolic guanylate cyclase from human placenta.

We have purified the soluble form of guanylate cyclase from human placenta greater than 2400-fold. The enzyme shared several characteristics with the enzyme purified from other sources including molecular mass and subunit composition, activation by divalent cations, inhibition by ATP and Michaelis constants. The enzyme, however, had a lower absorption maximum in the Soret region (417 +/- 1 nm) than the enzyme from other sources and was activated only one-fifth as much by nitric oxide as the bovine lung enzyme. It appears that the heme prosthetic group in the human placental enzyme may be hexa-coordinate and in the bovine lung enzyme the heme group may be penta-coordinate.     

gamma-Glutamyltransferase in human diploid fibroblasts and other mammalian cells.

gamma-Glutamyltransferase was determined in WI-38 human diploid fibroblasts and compared to enzyme levels determined in several other mammalian cell lines including: fibroblast-like cells from human skin, tibia and foreskin; epithelial-like cells from human, bovine and monkey kidney; and transformed cells (Chinese hamster ovary, HeLa S3 and SV-40 transformed WI-38). Transformed cells had the lowest activity found followed in increasing order by fibroblasts, human and bovine epithelial cells and monkey kidney epithelial cells. The enzyme isolated from the plasma membrane of WI-38 cells, like the enzyme from kidney and brain, was found to be irreversibly inhibited by iodoacetamide, reversibly by serine-borate, and had a strong specificity for certain amino acids. The possibility exists that gamma-glutamyltransferase could be involved in transport of amino acids into cells in culture; and glutamine, used in media, is an excellent substrate for the enzyme.     

Lens neutral endopeptidase occurs in other bovine and human tissues.

Lens neutral endopeptidase (EC 3.4.24.5) was previously thought to be unique to the eye lens. We report here the finding of a neutral endopeptidase, in a variety of bovine and human tissues, which is very similar both biochemically and immunologically to the lens endopeptidase. SDS/polyacrylamide-gel electrophoresis of partially purified enzyme fractions from various bovine tissues shows the characteristic pattern of at least eight bands with Mr values ranging from 24,000 to 32,000 which was described for the bovine-lens neutral endopeptidase. The relative activity of the enzyme varies from tissue to tissue with lung having the highest activity. Partially purified enzyme fractions from these tissues cross-react with antiserum raised in rabbit against bovine lens endopeptidase showing apparent identity when examined side by side in Ouchterlony double-diffusion tests. The human enzyme also cross-reacts with the antiserum but when tested by double-diffusion against the bovine enzyme the precipitin lines show spurring at the joining edges indicating a structural difference between the human and the bovine enzymes. It was also found by Western blot experiments, after denaturing polyacrylamide-gel electrophoresis of the enzyme, that the polypeptide components of the human and bovine enzymes show somewhat different banding patterns.     

Isolation and characterization of angiotensin converting enzyme from human kidney

Angiotensin converting enzyme [EC 3.4.15.1] was solubilized from the membrane fraction of human kidney cortex using trypsin and purified to homogeneity by DEAE-cellulose, hydroxylapatite and DEAE-Sephadex A-50 column chromatographies, preparative isoelectric focusing, and Sephadex G-200 gel filtration. The final recovery of the enzyme was 13.9%. The molecular weight of the enzyme was estimated to be 199,000 by a sedimentation equilibrium method. A value of 170,000 was obtained for the reduced and denatured enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was a glycoprotein consisting of a single polypeptide chain with an isoelectric point of 5.10. Neutral sugar accounted for 13% per weight of the enzyme. The purified enzyme had a specific activity of 96.9 mumol/min/mg protein for hippurylhistidylleucine. The Km value, Kcat value and hydrolytic coefficient (Kcat/Km) of the enzyme for hippurylhistidylleucine were 2.0 mM, 545 s-1 and 273 mM-1 . s-1, respectively. Rabbit antibody against the human kidney converting enzyme inhibited the activities of the enzymes from human lung and serum as equally as that from human kidney, but not those from sheep, dog, or rat sera. The human kidney and lung converting enzymes were immunologically identical on double immunodiffusion analysis.     

Isolation and properties of carboxycathepsin (peptidyl-dipeptidase) from bovine lung tissue]

2200-fold purified homogenous preparation of carboxycathepsin (peptidyl-dipeptidase) is isolated from bovine lung. The enzyme isolated converts angiotensine I into angiotensine II and distroys bradikinin. It is active in neutral medium, is activated by chloride ion and is inhibited by EDTA and Middle Asian snakes venom. The molecular weight of the enzyme is 180 000-190 000 as estimated by means of polyacrylamide gel electrophoresis, its isoelectric point is 4.48-4.53. The comparison of properties and specificity of carboxycathepsin from bovine lung and kidney draws to the conclusion that both enzymes are identical.     

Rapid affinity chromatographic purification of human lung and kidney angiotensin-converting enzyme with the novel N-carboxyalkyl dipeptide inhibitor N-[1(S)-carboxy-5-aminopentyl]glycylglycine

Human angiotensin-converting enzyme has been purified, in a single chromatographic step, using a novel N-carboxyalkyl dipeptide CA-GlyGly (N-[1(S)-carboxy-5-aminopentyl]glycylglycine) synthesised in our laboratory. CA-GlyGly is a weak competitive inhibitor, Ki = 0.18 mM, and its inhibitory profile is markedly pH-dependent. Human lung and kidney angiotensin-converting enzyme were solubilised with Triton X-100 and after ammonium sulphate fractionation the crude extract was applied to a column containing CA-GlyGly coupled to agarose via a 2.8 nm spacer group. Electrophoretically pure human angiotensin-converting enzyme could be eluted by raising the pH of the chromatography buffer from 7.50 to 9.50. The specific activity of human angiotensin-converting enzyme purified from lung was 104 units/mg, while that from kidney was 88 units/mg. Molecular weight for both enzymes was estimated to be 160,000. The Km with respect to hippuryl-L-histidyl-L-leucine was 1.9 mM in the case of lung angiotensin-converting enzyme and 1.7 mM in that of kidney angiotensin-converting enzyme, while for the substrate angiotensin I Km values were 62 microM and 76 microM, respectively. Hydrolysis of either substrate was chloride-dependent and both enzymes were strongly inhibited by captopril.     

Atrial natriuretic peptide binding, cross-linking, and stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity in cultured cells

The stimulation of cyclic GMP accumulation and particulate guanylate cyclase activity by atrial natriuretic peptide (ANP) was compared to the affinity and number of ANP receptors in eight cultured cell types. At 100 nM, ANP increased cyclic GMP by 13-fold in bovine adrenal cortical, 35-fold in human lung fibroblast, 58-fold in canine kidney epithelial, 60-fold in bovine aortic smooth muscle, 120-fold in rat mammary epithelial, 260-fold in rat Leydig, 300-fold in bovine kidney epithelial, and 475-fold in bovine aortic endothelial cells. ANP (1 microM) increased particulate guanylate cyclase activity by 1.5-, 2.5-, 3.1-, 3.2-, 5.0-, 7.0-, 7.8-, and 8.0-fold in bovine adrenal cortical, bovine aortic smooth muscle, human lung fibroblast, canine kidney epithelial, rat mammary epithelial, rat Leydig, bovine kidney epithelial, and bovine aortic endothelial cells, respectively. Specific 125I-ANP binding to intact rat Leydig (3,000 sites/cell; Kd = 0.11 nM), bovine aortic endothelial (14,000 sites/cell; Kd = 0.09 nM), bovine adrenal cortical (50,000 sites/cell; Kd = 0.12 nM), human lung fibroblast (80,000 sites/cell; Kd = 0.32 nM), and bovine aortic smooth muscle (310,000 sites/cell; Kd = 0.82 nM) cells was saturable and high affinity. No specific and saturable ANP binding was detected in bovine and canine kidney epithelial and rat mammary epithelial cells. Two ANP-binding sites of 66,000 and 130,000 daltons were specifically labeled by 125I-ANP after cross-linking with disuccinimidyl suberate. The 130,000-dalton ANP-binding sites bound to a GTP-agarose affinity column, and the specific activity of guanylate cyclase was increased by 90-fold in this fraction. Our results demonstrate that the increase in cyclic GMP accumulation and particulate guanylate cyclase activity by ANP does not correlate with the affinity and number of ANP-binding sites. These results suggest that multiple populations of ANP receptors exist in these cells and that only one receptor subtype (130,000 daltons) is associated with particulate guanylate cyclase activity.     

Expression of antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia coli

His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multiners. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3 mg/l and analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2 mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.     

Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins.

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.     

Facile incorporation of urea pseudopeptides into protease substrate analogue inhibitors

A new procedure that employs a one-pot, oxidative Hofmann rearrangement to incorporate a urea linkage into peptide backbones is detailed herein. This methodology was used to replace the scissile peptide bonds of [Leu5]enkephalin and a hexapeptide HIV-1 protease substrate. The [Leu5]enkephalin analogue was found to inhibit cleavage of hippurylhistidylleucine (HHL) by porcine kidney angiotensin-converting enzyme (PK-ACE) with a 0.88 mM IC50 value, comparable to the Michaelis constant of [Leu5]enkephalin with the same enzyme. The HIV-1 protease substrate analogue was shown to inhibit HIV-1 protease with an IC50=34 microM.     

Histochemical and immunological demonstration of purple acid phosphatase in human and bovine alveolar macrophages

A tartrate-resistant purple acid phosphatase was localized in human and bovine alveolar macrophages by enzyme- and immuno-histochemistry using an antibody to bovine spleen purple phosphatase. The enzyme could be detected in human and bovine lung tissues as well as on cytospin preparations of alveolar macrophage suspensions from bronchoalveolar lavages. The immunological identity of human and bovine purple phosphatases from alveolar macrophages was demonstrated by Western blot analysis of material separated by polyacrylamide gel electrophoresis. A possible significance of the purple phosphatase as a marker enzyme of activated cells of the mononuclear phagocyte system is discussed.     

Effects of singlet oxygen on the extracellular matrix protein collagen: oxidation of the collagen crosslink histidinohydroxylysinonorleucine and histidine

The reaction of singlet oxygen, a putative agent of skin photodamage, with the dermal collagen crosslink histidinohydroxylysinonorleucine (HHL) and its precursor histidine is reported. Reaction studies were performed with both purified HHL and bovine dermal tissue. We demonstrate that singlet oxygen can selectively oxidize HHL and histidine amino acid residues in dermal tissue and that intermediate oxidation products of histidine lead to new crosslink products. A novel mechanism for crosslink formation was proposed to involve nucleophilic addition to a transient imidazolone intermediate formed from singlet oxygen oxidation of the histidine imidazole moiety. The implication for such adduct formation and histidine oxidation in collagen proteins is the expression of aberrant collagen crosslinks, perturbation of the dermal collagen function, and hence an altered dermal state.     

Histochemical and immunological demonstration of purple acid phosphatase in human and bovine alveolar macrophages

Summary A tartrate-resistant purple acid phosphatase was localized in human and bovine alveolar macrophages by enzyme- and immuno-histochemistry using an antibody to bovine spleen purple phosphatase. The enzyme could be detected in human and bovine lung tissues as well as on cytospin preparations of alveolar macrophage suspensions from bronchoalveolar lavages. The immunological identity of human and bovine purple phosphatases from alveolar macrophages was demonstrated by Western blot analysis of material separated by polyacrylamide gel electrophoresis. A possible significance of the purple phosphatase as a marker enzyme of activated cells of the mononuclear phagocyte system is discussed.