Interaction between leukocytic elastase and Kunitz-type inhibitors from bovine spleen

The four Kunitz-type protease inhibitors purified from bovine spleen, which include the basic pancreatic trypsin inhibitor (BPTI), form stable complexes with human leukocytic elastase. The values of the affinity constants of these complexes are similar, in agreement with the great structural similarity of the four inhibitors, but are lower than those measured for the complexes with other serine proteases. Two main factors appear to be responsible for the stability of these complexes, i.e., hydrophobic interactions and ionization phenomena that take place during complex formation. These two factors have been analyzed in terms of the general model previously used for describing the interaction between the serine proteases and their natural inhibitors.     

Primary structure and antiproteolytic activity of a Kunitz-type inhibitor from bovine spleen

The amino acid sequence of protease inhibitor II, previously isolated from bovine spleen, has been completely elucidated and reveals a high homology (approximately 90%) with that of bovine pancreatic trypsin inhibitor (BPTI), the well-known Kunitz inhibitor. The secondary and tertiary structure of this new inhibitor appears similar to that of BPTI. Whereas its affinity for bovine trypsin, chymotrypsin, and trypsinogen is almost identical to that of BPTI, the affinity for porcine pancreatic kallikrein is decreased, as expected on the basis of the amino acid substitutions. Analysis of the pH dependence of the affinity constant confirms the previous assignment of the ionizable groups, whose pK values are perturbed on complex formation, to kallikrein and not to the inhibitor molecule.     

Kunitz-type protease inhibitors from acrorhagi of three species of sea anemones

Sea anemones are rich in biologically active polypeptides such as toxins and protease inhibitors. These polypeptides have so far been isolated from whole bodies, tentacles or secreted mucus. Recently, two novel peptide toxins with crab lethality have been isolated from acrorhagi (specialized aggressive organs elaborated by only certain species of sea anemones belonging to the family Actiniidae) of Actinia equina. This prompted us to survey biologically active polypeptides in the acrorhagi of two species of sea anemones, Anthopleura aff. xanthogrammica and Anthopleura fuscoviridis. No potent crab lethality was displayed by the acrorhagial extracts of both species. However, significantly high protease inhibitory activity was instead detected in the acrorhagial extracts of the two species and also in that of A. equina. From the acrorhagi of A. equina, A. aff. xanthogrammica and A. fuscoviridis, one (AEAPI), one (AXAPI) and two (AFAPI-I and AFAPI-III) protease inhibitors were isolated, respectively. The complete amino acid sequences of the four inhibitors were elucidated by N-terminal sequencing and sequencing of the C-terminal peptide fragment produced upon asparaginylendopeptidase digestion. The determined amino acid sequences revealed that all the four inhibitors are new members of the Kunitz-type protease inhibitor family.     

Isolation of three types of Gi from bovine spleen

We have previously reported the purification of two alpha subunits of G proteins, Gi2 and Gi3, from bovine spleen. However, it recently became clear that the preparation of Gi3 alpha contained a significant amount of Gi1 alpha by the immunoblot analysis using specific antibodies. In this study, we purified these G proteins as a trimer form from bovine spleen, and obtained following results. (1) Gi3 was separated from Gi1 using Mono Q column chromatography. Isoelectric focusing was employed to distinguish Gi3 from Gi1 in the column eluates. (2) Purified Gi2 and Gi3 retained much higher activities to bind GTP gamma S or to be ADP-ribosylated by pertussis toxin than the alpha subunits purified previously. (3) Using these spleen Gi2 and Gi3 and bovine brain Gi1, the parameter of GTP gamma S binding to the three types of Gi was compared. Three Gis showed different rates of GTP gamma S binding but showed the similar Kd values.     

Immunochemical studies on the Kunitz type inhibitors from bovine spleen

Specific immunoglobulins for bovine spleen inhibitor IV, which is identical to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) from bovine lung, were purified from the serum of immunized rabbits. Immunological and immunochemical experiments have shown that the four inhibitors previously isolated from bovine spleen are cross-reacting antigens with the anti-inhibitor IV - antiserum; however, part of the antibodies are precipitated by inhibitors I, II and III, whereas the remaining ones are only specific for the antigenic determinants present on the inhibitor IV molecule.     

Kunitz-type protease inhibitors group B from Solanum palustre

Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.     

Kunitz型丝氨酸蛋白酶抑制剂研究进展

Kunitz型丝氨酸蛋白酶抑制剂是一类普遍存在的蛋白酶抑制剂,在体内各项生命活动中扮演着重要角色。这类抑制剂结构稳定且富有特色,通常具有一个或几个串联存在的Kunitz结构域,能够以类似底物的方式与丝氨酸蛋白酶结合,从而抑制酶的活性。在功能方面,Kunitz型丝氨酸蛋白酶抑制剂参与凝血和纤维蛋白溶解、肿瘤免疫、炎症调节以及抵抗细菌、真菌感染等过程。文中就Kunitz型丝氨酸蛋白酶抑制剂研究进展作一综述,为新型Kunitz型丝氨酸蛋白酶抑制剂的开发提供研究思路。     

Monoclonal antibodies to three phospholipase C isozymes from bovine brain

Murine hybridoma cell lines secreting antibodies against the three bovine isozymes of phosphoinositide-specific phospholipase C (PLC) were established: 6, 23, and 12 lines were obtained for PLC-I (150 kDa), PLC-II (145 kDa), and PLC-III (85 kDa), respectively. The antibodies were purified from ascites fluid, and their properties were studied in detail. All the antibodies cross-reacted with their corresponding PLC enzymes, but not with the other two isozymes, suggesting that the three enzymes contain very different antigenic determinants. The six antibodies elicited by bovine PLC-I also cross-reacted with human and rat enzyme, whereas three each from anti-PLC-II antibodies and anti-PLC-III antibodies did not react with the enzymes from different species. Each antibody exerts different effects on the phosphatidylinositol-hydrolyzing activity of PLC. The most inhibitory antibody for either isozyme PLC-I or PLC-II exhibits 80% inhibition, whereas no more than 20% inhibition was observed for the anti-PLC-III antibodies. Purified PLC-I frequently contains catalytically active 140- and 100-kDa forms and an inactive 41-kDa protein in addition to the intact 150-kDa form, probably due to its high sensitivity to an unidentified endogenous protease. The five anti-PLC-I antibodies which bind to the denatured 150-kDa polypeptide also recognized the 140-kDa form, whereas only three cross-reacted with the 100-kDa form, and the remaining two bound to the 41-kDa protein. Competitive binding studies with intact PLC enzymes and Western blot experiments with proteolytic digests revealed that the 6 anti-PLC-I, 23 anti-PLC-II, and 12 anti-PLC-III antibodies bind at least five, six, and seven different epitopes on PLC-I, PLC-II, and PLC-III, respectively. The fact that these monoclonal antibodies bind to different epitopes on the same enzyme allowed one to develop a highly specific and sensitive tandem radioimmunoassay for quantitating PLC-I, PLC-II, and PLC-III. The principle of the assay is that binding of an 125I-labeled antibody to the antigen immobilized by another antibody at a distinctive binding site is proportional to the amount of antigen present. By using this method, PLC-I, PLC-II, and PLC-III could be measured quantitatively in the presence of other proteins, detergents, lipids, polyanions, and metal ions, all of which greatly affect the activity of PLC enzymes.     

Characterization of a set of antibodies specific for three human histone H1 subtypes

A series of human histone H1 subtype-specific antibodies are described that were generated for localization and functional studies. Since our previous attempts to produce such antibodies against intact subtypes met with limited success, resulting in one antibody against a subtype we have designated H1-3, the approach used in the work presented is based on the production of antibodies against synthetic peptides or peptide fragments encompassing the variant NH₂-terminal region of cach protein. Subtype-specific antibodies were obtained against synthetic peptides derived from subtypes designated H1-1 and H1-2 and the NH₂-terminal fragment from an N-bromosuccinimide digest of H1–4. Antibody specificities were documented in all cases by enzymelinked immunosorbent and protein immunoblot assays against the purified subtypes as well as immunoblots against whole cell and nuclear extracts. In addition, the in vivo distribution of each antibody was determined by indirect immunofluorescence. H1-1 appears to be distributed in parallel with DNA concentration, similar to the results with an antibody that recognizes all subtypes, However, H1–2 and H1–4 are non-uniformly distributed, exhibiting similar punctate staining patterns. The staining patterns described are different from the pattern desciribed for the distribution of H1–3, suggesting that several subtypes are concentrated in distinct regions of the nucleus and, therefore, may be associated with distinct regions of the genome.     

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, VII. Characterization of the bovine inhibitor as double-headed trypsin-elastase inhibitor

The acid-resistant 14-kDa inhibitor BI-14, released from bovine inter-alpha-trypsin inhibitor, consists of two tandem Kunitz-type domains, and is of a double-headed nature. The Arg-Thr bond connecting both domains was cleaved and the two inhibitory domains were separated. The N-terminal domain is an inhibitor of bovine chymotrypsin and elastases from porcine pancreases and human polymorphonuclear granulocytes, whereas the C-terminal domain interacts with trypsin, plasmin, and chymotrypsin. In the intact inhibitor BI-14 both domains interact independently with the proteinases.     

Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross-react with other metabolites. These methylsulfinylalkyl glucosinolate specific antibodies have prospective use in multiple applications such as ELISA, co-immunoprecipitation and immunolocalization of glucosinolates.     

Functional comparison of homologous members of three groups of Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

For functional studies, nine cDNAs encoding Kunitz-type enzyme inhibitors from potato tubers were expressed as GST (glutathione S transferase)-tagged fusion proteins in the fission yeast Schizosaccharomyces pombe. The inhibitors represented the three major homology groups A, B and C found in tubers. Members of the same homology group were at least 90% identical in sequence. The purified GST fusion proteins were tested for their ability to inhibit the proteases trypsin, alpha-chymotrypsin, subtilisin, papain and aspergillopepsin I, and for inhibition of the growth of fungi. Fusion proteins belonging to the same and different homology groups were found to exhibit distinct protease inhibition profiles. Removal of the GST tag by cleavage with enterokinase did not change the inhibition profile but increased the inhibitory activity. Group A and B inhibitors affected the proteases to different extents, whereas group C inhibitors showed only weak or no protease inhibition. One fusion protein completely inhibited aspergillopepsin I. One fusion protein each of groups A and B strongly inhibited mycelial growth of the fungus Fusarium moniliforme. The results suggest functional polymorphism among closely related members of the Kunitz-type inhibitor family.     

Structural diversity and organization of three gene families for Kunitz-type enzyme inhibitors from potato tubers ( Solanum tuberosum L.)

In the potato, Kunitz-type enzyme inhibitors are abundant and highly polymorphic small proteins found in tubers. DNA sequence analysis of 1596 unselected ESTs (expressed sequence tags) from mature tubers of the cultivars Provita and Saturna resulted in the identification of 55 different DNA sequences with high sequence similarity to Kunitz-type enzyme inhibitors. The frequency of Kunitz-type inhibitor ESTs in Provita was four times higher than in Saturna tubers, and none of the Provita ESTs was identical to any of the Saturna ESTs. A phenogram constructed from the deduced amino acid sequences of the inhibitors revealed three major homology groups-A, B and C. Group A inhibitors were all derived from Provita ESTs. Inhibitor groups A and B were more similar to each other than to group C inhibitors, and for most members within-group similarity was at least 90%. Non-conservative amino acid substitutions and insertion/deletion polymorphisms suggest functional differentiation between members of the gene family. A minimum of 21 genes for Kunitz-type enzyme inhibitors (six for group A, nine for group B and six for group C) was estimated to exist in the potato genome. Genetic mapping and the identification of BAC (bacterial artificial chromosome) clones containing more than one member of the gene family indicated that most inhibitor genes of groups A, B and C are organized in a cluster that maps to a single region on potato chromosome III.     

Bauhinia Kunitz-type proteinase inhibitors: structural characteristics and biological properties

Abstract Plant proteinase inhibitors are involved in the regulation of the activity of many proteinases and, in consequence, in biological processes driven by proteolysis. In this review, we summarize recent results on the activity of native Bauhinia inhibitors and synthetic derivatives. Structural and functional characteristics and the potential therapeutic use of these inhibitors are also discussed.     

Characterization of a new bioactive protein from bovine seminal fluid

A new acidic seminal fluid protein (aSFP) was purified from bovine seminal fluid, using anion exchange chromatography and FPLC on MonoQ. The purified aSFP displays a pI of 4.8 and an apparent molecular weight of 14 kDa. Homogeneity of aSFP was demonstrated by FPLC and SDS-polyacrylamide gel electrophoresis. Monospecific anti-aSFP IgGs were employed to characterize aSFP in bovine seminal plasma and seminal vesicle secretion by immuno blot analysis. Proteinchemical characterization of aSFP included amino acid analysis as well as determination of 23 amino acid residues of the N-terminal sequence of aSFP. According to this sequence, aSFP appears to represent a hitherto unknown protein. aSFP stimulated cell division and progesterone secretion of bovine granulosa cells in vitro in a potent and dose dependent manner. aSFP appears to be a potent growth factor with effects on ovarian granulosa cells.