Identification of a rat liver dipeptidyl aminopeptidase IV with a liver plasma membrane glycoprotein (gp110). A study using dipeptidyl aminopeptidase IV-deficient rats.

A rat liver plasma membrane glycoprotein, gp110, was compared with dipeptidyl aminopeptidase IV (DAP IV) by using Wistar rats (DAP IV-positive rats) and Fischer 344 rats (DAP IV-negative rats). Fischer rats also lacked gp110 and gp110 of Wistar rats had DAP IV activity. Furthermore, we showed that the C-terminal sequence of gp110 was Ser-Leu-Arg, which was the same as the C-terminal amino acid sequence deduced from the nucleotide sequence of the cDNA of DAP IV. According to these results, we concluded that gp110 was identical with DAP IV.

Is CD26/dipeptidyl peptidase IV a really important molecule in T cell activation of a certain rat strain?

A new monoclonal antibody (MS-7 mAb) was raised to investigate the real role of the membrane-associated molecule CD26/dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5), which transduces activation signals in T cells. A strain of rats which is deficient in DPP IV was used. MS-7 mAb recognized DPP IV (110 kDa) and its 60 kDa fragment, starting at the 281st residue corresponding to the extracellular one comprising the active-site sequence Gly-X-Ser631-X-Gly of DPP IV. MS-7 mAb recognized CD26 on T cells of DPP IV+ rats both before and after mitogen activation. CD26 expression and DPP IV enzyme activity are increased on T cells following their activation; nevertheless, no CD26 was expressed on T cells of DPP IV- rats, and no DPP IV enzyme activity was detected either before or after mitogen activation. In addition, MS-7 mAb inhibited the mitogen-stimulated proliferation of DPP IV+ rats, but did not affect that of DPP IV- rats. These results suggest that CD26/DPP IV is not a necessary molecule in T cell activation, and that there is some other bypass in T cell activation of DPP IV- rats.

Dipeptidyl peptidase IV from human serum: purification, characterization, and N-terminal amino acid sequence.

Dipeptidyl peptidase IV (DPP IV) in normal human serum was purified 14,400-fold with a 25% yield to homogeneity. The molecular weight of the purified enzyme was approximately 110,000 on SDS-PAGE, almost the same as that of human kidney membrane-bound DPP IV. No difference was found between the two enzymes enzymologically and immunologically, either in substrate specificity, susceptibility to inhibitors, or cross-reactivity with an anti-rat kidney DPP IV antibody, or in their ability to bind adenosine deaminase. However, the N-terminal amino acid sequence of serum DPP IV lacked the transmembrane domain of the membrane-bound enzyme and started at the 39th position, serine, from the N-terminus predicted from the cDNA nucleotide sequence. These results suggest that membrane-bound DPP IV loses its transmembrane domain upon release into the serum, and that its structure on the plasma membrane is not required for its binding to adenosine deaminase.

Comparison of the abilities of proteins from Bartonella bacilliformis and Bartonella henselae to deform red cell membranes and to bind to red cell ghost proteins.

Infections in humans by Bartonella bacilliformis, but not Bartonella henselae, are characterized by invasion of red cells. Supernatants of culture medium from B. bacilliformis and B. henselae each contain a protein which causes invagination of membranes of human red cells and formation of intracellular vacuoles. These two proteins are very similar in molecular mass, heat stability and mechanism of action. B. henselae does not bind to human red cells, but human red cell ghost membrane proteins were recognized by both bacteria, five by B. bacilliformis and the same five, and one additional protein by B. henselae. Two of these proteins had molecular masses consistent with actin and spectrin. Actin binds to five electroblotted outer membrane proteins from B. henselae and four of these proteins are retained on an actin-Sepharose column.

Ageing in Werner syndrome.

Oxidative stress markers including pentosidine and homocysteine were examined comparing them with inflammation markers including highly sensitive C-reactive protein (hsCRP) and matrix metalloproteinase-9 (MMP-9) in serum from patients with Werner syndrome (WS) and healthy individuals. Elevation of serum pentosidine correlated significantly with normal aging in healthy individuals (p < 0.0004). Serum pentosidine in WS increased significantly compared with age-matched healthy individuals (p < 0.05). Serum homocysteine levels increased insignificantly with normal aging in healthy individuals and in WS compared with age-matched healthy individuals. As both pentosidine and homocysteine levels did not correlate with hsCRP or MMP-9, both oxidative stress markers may be differentially regulated by inflammation.

Correlations between matrix metalloproteinase-9 and adenosine deaminase isozymes in synovial fluid from patients with rheumatoid arthritis.

OBJECTIVE: To clarify the significance of increased adenosine deaminase (ADA) isozyme activities in synovial fluid (SF) from patients with rheumatoid arthritis (RA). METHODS: ADA isozyme activities were measured using colorimetric assays. Concentrations of matrix metalloproteinase-9 (MMP-9) were measured by ELISA. RESULTS: Levels of ADA isozyme activities in RA SF were significantly higher than those from patients with osteoarthritis and patients with traumatic injuries. Significant positive correlations between MMP-9 concentration and ADA activities were observed in RA SF (MMP-9 vs total ADA: r = 0.880; vs ADA1: r = 0.829; vs ADA2: r = 0.823; p < 0.001). CONCLUSION: Elevated levels of ADA activities were found in SF from patients with RA. There were significant positive correlations between MMP-9 and ADA isozymes. These results may reflect the inflammatory condition of patients with RA.

N-terminal amino acid sequence of the 60-kDa protein of rat kidney dipeptidyl peptidase IV.

The N-terminal amino acid sequence of the 60-kDa protein of purified dipeptidyl peptidase IV (DPP IV) was determined. The protein was isolated from rat kidney by detergent solubilization. The first 22 amino acids were sequenced; these matched the predicted sequence between residues 281 and 302 of the amino-terminal region of rat liver DPP IV.

CD26/dipeptidyl peptidase IV does not work as an adenosine deaminase-binding protein in rat cells.

In this paper, we describe further characterization of the membrane-associated molecule CD26/dipeptidyl peptidase IV (DPP IV), which is said to be adenosine deaminase-binding protein (ADA-bp) in humans, to clarify its association with ADA in rat immune cells. For this purpose, we used three types of rats: DPP IV+ rats; DPP IV- rats, which lack enzyme activity and immunological reactivity of DPP IV; and ADA- rats, which have reduced ADA activity due to continuous peritoneal injection of 2'-DCF, a potent inhibitor of ADA. ADA existed in the cells of DPP IV+ and DPP IV- rats, but it did not exist on the cell surface in either rat. ADA- rats showed a decrease in ADA activity and in the number of immune cells, but no effect on DPP IV was observed. These data suggest that in rats, in contrast to humans, DPP IV does not exist as ADA-bp.

Identification of an alanine aminopeptidase in human maternal serum as a membrane-bound aminopeptidase N.

In addition to cystine aminopeptidase (oxytocinase) alanine aminopeptidase is present at high levels in the serum of pregnant women. In this study we compared the enzyme with membrane-bound aminopeptidase N purified from human placenta. Comparison of catalytic and immunological properties and N-terminal sequence analyses revealed that the enzymes were differentially processed derivatives of the same protein, and that the N-terminal 68 residues of aminopeptidase N were deleted in the alanine aminopeptidase. The deleted sequence contains a small cytoplasmic region, a hydrophobic transmembrane domain and a junctional domain. These results suggest that the enzyme may be released into the maternal circulation as a result of lacking these three domains.

Increased excretion of proline-containing peptides in dipeptidyl peptidase IV-deficient rats.

We have reported that a substrain of Fischer 344 rat exhibits a deficiency of dipeptidyl peptidase IV (Watanabe et al. Experientia 43: 400-401, 1987). In this study, this rat model was found to excrete large amounts of proline-containing peptides in the urine. The peptide fraction also contained other amino acids, such as hydroxyproline and glycine. The results indicate that the enzyme might be involved in the metabolism of collagens and related peptides.

Induction of a novel gelatinolytic activity in synovial tissue of patients with rheumatoid arthritis.

Gelatinolytic activity induced by longtime incubation at 37 degrees C was found in extracts of rheumatoid synovial tissues. The enzyme activity was assessed by gelatin zymography and 3H-gelatin degradation assay. The observed enzyme had different characteristics from matrix metalloproteinases; it did not require metal ions for activity. However, metallocheltors blocked activation and addition of some metal ions restored the activation. The molecular size of the enzyme was changed time-dependently. The approximate molecular weight of the first enzyme produced by incubation was 65 kDa and it was converted to a broad size molecule with a molecular weight of 50 kDa after further incubation. Substrate specificity was detected for denatured collagen types I, II, III and IV.

An antibody specific to N-terminal amino acid sequence of human maternal serum cystine aminopeptidase and its applications.

An antibody directed against a synthetic peptide corresponding to the N-terminal sequence of human cystine aminopeptidase (CAP) present in maternal serum was prepared. Although the antibody did not immunoprecipitate the activity of CAP, it was useful for purification and immunoblot analysis of CAP protein. An antipeptide antibody-conjugated Sepharose 4B column was very effective in purifying a single CAP protein from partially purified enzyme preparation, and Western blotting confirmed the binding of the antibody to CAP protein.

Aminopeptidase N in sera of healthy subjects is a different N-terminal processed derivative from the one obtained from maternal serum.

A major aminopeptidase present in normal human serum was purified to homogeneity as a 150-kDa molecular species. Western blotting confirmed the binding of an anti-aminopeptidase N antibody to the protein. The N-terminal amino acid sequence of the enzyme was determined. The first 13 amino acids of the enzyme completely matched amino acids 59-71 of the sequence predicted from the human intestinal aminopeptidase N cDNA nucleotide sequence. As reported previously, aminopeptidase N from maternal serum had 68 fewer amino acid residues at the N-terminus than the enzyme obtained from detergent-solubilized membranes. The results indicate that aminopeptidase N in normal serum is a different N-terminal processed derivative from that obtained from maternal serum.