scholarly journals Fragmentation of human polymorphonuclear-leucocyte collagenase

1993 ◽  
Vol 291 (3) ◽  
pp. 847-854 ◽  
Author(s):  
V Knäuper ◽  
A Osthues ◽  
Y A DeClerck ◽  
K E Langley ◽  
J Bläser ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Sequence Data ◽  
Enzymic Activity ◽  
Polymorphonuclear Leucocyte ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Molar Ratio ◽  
Terminal Sequence ◽  
Activity Profile ◽  
Binding Reaction ◽  
Active Collagenase

Human polymorphonuclear-leucocyte collagenase (M(r) 64,000) shows autoproteolytic degradation to two major fragments of M(r) 40,000 and M(r) 27,000. N-terminal sequence data and investigation of the substrate specificity of the fragments demonstrate that the M(r)-40,000 fragment corresponds to the catalytic domain, whereas the M(r0-27,000 fragment shows no enzymic activity. The activity profile of the M(r)-40,000 fragment is comparable with the specificity of the intact active collagenase (M(r) 64,000), but the ability to cleave collagen was lost. The enzymic activity of this fragment can be inhibited by either tissue inhibitor of metalloproteinase (TIMP)-1 or recombinant TIMP-2 in a 1:1 molar ratio. The C-terminal part of the enzyme (M(r) 27,000), important for the binding reaction with collagen substrates, is involved in collagenolysis.

scholarly journals Expression of human recombinant 72 kDa gelatinase and tissue inhibitor of metalloproteinase-2 (TIMP-2): characterization of complex and free enzyme

1993 ◽  
Vol 289 (2) ◽  
pp. 411-416 ◽  
Author(s):  
R Fridman ◽  
R E Bird ◽  
M Hoyhtya ◽  
M Oelkuct ◽  
D Komarek ◽  
...  
Keyword(s):  
Specific Activity ◽  
Expression System ◽  
Enzymic Activity ◽  
Free Enzyme ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Molar Ratio ◽  
Collagen Type Iv ◽  
Tissue Inhibitor ◽  
Type Iv Collagenase ◽  
Type Iv

The human 72 kDa gelatinase/type IV collagenase is a metalloproteinase that is thought to play a role in metastasis and angiogenesis. The 72 kDa progelatinase can be isolated from conditioned media as a complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2). To investigate 72 kDa gelatinase-TIMP-2 interactions and to compare the activity of the complex versus that of the free enzyme, we have expressed and purified human 72 kDa progelatinase and TIMP-2 as single proteins in a recombinant vaccinia virus mammalian cell expression system. The recombinant 72 kDa progelatinase was able to bind TIMP-2, and it digested gelatin and collagen type IV after activation by p-aminophenylmercuric acid (APMA). The specific activity of the recombinant free enzyme was 20-fold higher than the activity of an APMA-treated stoichiometric complex of recombinant 72 kDa progelatinase and TIMP-2. Also, TIMP-2 caused an 86% inhibition of activity when added to the activated enzyme at a 1:1 molar ratio. Activation of the free recombinant 72 kDa progelatinase yielded the 62 kDa species and two fragments of 46 and 35 kDa that cross-reacted with monoclonal antibodies to the 72 kDa proenzyme. TIMP-2 inhibited the conversion of the recombinant proenzyme to the 62 kDa species and the appearance of the 45 and 35 kDa bands. These results suggest that TIMP-2 is not only a potent inhibitor of the activated enzyme but also prevents the generation of low-molecular-mass species and full enzymic activity from the zymogen.

scholarly journals Arabinanase A from Pseudomonas fluorescens subsp. cellulosa exhibits both an endo- and an exo- mode of action

1997 ◽  
Vol 323 (2) ◽  
pp. 547-555 ◽  
Author(s):  
Vincent A. McKIE ◽  
Gary W. BLACK ◽  
Sarah J. MILLWARD-SADLER ◽  
Geoffrey P. HAZLEWOOD ◽  
Judith I. LAURIE ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Mode Of Action ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Single Copy ◽  
Terminal Sequence ◽  
Reading Frame ◽  
Significant Release

Pseudomonas fluorescens subsp. cellulosa expressed arabinanase activity when grown on media supplemented with arabinan or arabinose. Arabinanase activity was not induced by the inclusion of other plant structural polysaccharides, and was repressed by the addition of glucose. The majority of the Pseudomonas arabinanase activity was extracellular. Screening of a genomic library of P. fluorescens subsp. cellulosa DNA constructed in Lambda ZAPII, for recombinants that hydrolysed Red-dyed arabinan, identified five arabinan-degrading plaques. Each of the phage contained the same Pseudomonas arabinanase gene, designated arbA, which was present as a single copy in the Pseudomonas genome. The nucleotide sequence of arbA revealed an open reading frame of 1041 bp encoding a protein, designated arabinanase A (ArbA), of Mr 39438. The N-terminal sequence of ArbA exhibited features typical of a prokaryotic signal peptide. Analysis of the primary structure of ArbA indicated that, unlike most Pseudomonas plant cell wall hydrolases, it did not contain linker sequences or have a modular structure, but consisted of a single catalytic domain. Sequence comparison between the Pseudomonas arabinanase and proteins in the SWISS-PROT database showed that ArbA exhibits greatest sequence identity with arabinanase A from Aspergillus niger, placing the enzyme in glycosyl hydrolase Family 43. The significance of the differing substrate specificities of enzymes in Family 43 is discussed. ArbA purifed from a recombinant strain of Escherichia coli had an Mr of 34000 and an N-terminal sequence identical to residues 32–51 of the deduced sequence of ArbA, and hydrolysed linear arabinan, carboxymethylarabinan and arabino-oligosaccharides. The enzyme displayed no activity against other plant structural polysaccharides, including branched sugar beet arabinan. ArbA produced almost exclusively arabinotriose from linear arabinan and appeared to hydrolyse arabino-oligosaccharides by successively releasing arabinotriose. ArbA and the Aspergillus arabinanase mediated a decrease in the viscosity of linear arabinan that was associated with a significant release of reducing sugar. We propose that ArbA is an arabinanase that exhibits both an endo- and an exo- mode of action.

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A

2001 ◽  
Vol 359 (3) ◽  
pp. 497-505 ◽  
Author(s):  
Sunke HIMPEL ◽  
Pascal PANZER ◽  
Klaus EIRMBTER ◽  
Hanna CZAJKOWSKA ◽  
Muhammed SAYED ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Molecular Model ◽  
Enzymic Activity ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Family ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Protein kinases of the DYRK (‘dual-specificity tyrosine-regulated kinase’) family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319–Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

Sequence, expression and localization of calmodulin-domain protein kinases inEimeria tenellaandEimeria maxima

Parasitology ◽  
1996 ◽  
Vol 113 (5) ◽  
pp. 439-448 ◽  
Author(s):  
P. P. J. Dunn ◽  
J. M. Bumstead ◽  
F. M. Tomley
Keyword(s):  
Host Cell ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Sequence Data ◽  
Host Cells ◽  
Cdna Clones ◽  
Host Cell Membrane ◽  
Amino Terminal ◽  
Domain Protein

SUMMARYWe have isolated and sequenced cDNA clones fromEimeria tenellaandEimeria maximawhich encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains – an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 ‘EF-hand’ motifs. Expression of theE. tenellacalmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells inin vitroculture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells byE. tenella.

scholarly journals Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain

1993 ◽  
Vol 295 (1) ◽  
pp. 273-276 ◽  
Author(s):  
A J Fosang ◽  
K Last ◽  
V Knäuper ◽  
P J Neame ◽  
G Murphy ◽  
...  
Keyword(s):  
Human Fibroblast ◽  
Catalytic Domain ◽  
Polymorphonuclear Leucocyte ◽  
Gelatinase A ◽  
Cleavage Sites ◽  
Similar Extent ◽  
Human Sequence ◽  
Fibroblast Collagenase ◽  
Gelatinases A And B

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase

2004 ◽  
Vol 32 (5) ◽  
pp. 764-765 ◽  
Author(s):  
O. Plíhal ◽  
J. Sklenář ◽  
J. Kmoníčková ◽  
P. Man ◽  
P. Pompach ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Enzymic Activity ◽  
Enzyme Activation ◽  
Protein Architecture ◽  
Fungal Enzyme ◽  
Hexa Gene ◽  
Catalytic Unit ◽  
Complex Protein ◽  
The Stability ◽  
Inhibition Studies

β-N-Acetylhexosaminidase from a filamentous fungus Aspergillus oryzae is a secreted enzyme known to be an important component of the binary chitinolytic system. Cloning of the hexA gene and sequencing of the enzyme revealed its unique preproprotein structure. While the enzyme's zincin-like and catalytic domain had significant similarities with members of the glycohydrolase 20 family, the propeptide was unique for the fungal enzyme. Detailed pulse–chase and inhibition studies revealed that propeptide was processed during the biosynthesis of the enzyme. Moreover, the presence of propeptide was necessary for enzyme activation, dimerization and secretion. The catalytic unit was N-glycosylated, and the propeptide was O-glycosylated, both in their C-terminal parts. Deglycosylation experiments revealed that the N-glycosylation increased the stability and solubility of the enzyme. In contrast, O-glycosylated propeptide was necessary to attain the full enzymic activity.

scholarly journals Purification and N-terminal sequence of the p21rho GTPase-activating protein, rho GAP

1991 ◽  
Vol 276 (3) ◽  
pp. 833-836 ◽  
Author(s):  
M D Garrett ◽  
G N Major ◽  
N Totty ◽  
A Hall
Keyword(s):  
Molecular Mass ◽  
Binding Proteins ◽  
Enzymic Activity ◽  
Gtpase Activity ◽  
Ras Proteins ◽  
Terminal Sequence ◽  
Human Spleen ◽  
Gtp Binding Proteins ◽  
Gtpase Activating Proteins ◽  
Gtp Binding

Eukaryotic cells contain numerous small-molecular-mass GTP-binding proteins, but the processes that they regulate are not known. Different members of this protein family appear to be associated with specific GTPase-activating proteins (GAPs), and we have previously reported the identification of a cytoplasmic GAP (rho GAP) that stimulates the GTPase activity of p21rho but not of other small-molecular-mass GTP-binding proteins. We have now purified rho GAP 2000-fold from human spleen tissue using f.p.l.c. Electrotransfer of this 27.5 kDa protein on to an Immobilon-P transfer membrane followed by reconstitution of its enzymic activity confirmed its identity. Rho GAP was subjected to N-terminal sequence analysis and 15 amino acids were obtained. The sequence showed 53% identity with a region present in IRA1, a protein which stimulates the GTPase activity of RAS proteins in Saccharomyces cerevisiae. These results suggest that there is a family of sequence-related GAP proteins, which to date includes ras GAP and its yeast counterparts IRA1 and IRA2, rho GAP and the Neurofibromatosis gene product NF1.

scholarly journals A tobacco (Nicotiana tabaccum) calmodulin-binding protein kinase, NtCBK2, is regulated differentially by calmodulin isoforms

2003 ◽  
Vol 376 (1) ◽  
pp. 291-302 ◽  
Author(s):  
Wei HUA ◽  
Shuping LIANG ◽  
Ying-Tang LU
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Dissociation Constants ◽  
Binding Protein ◽  
Enzymic Activity ◽  
Protein Motif ◽  
Independent Manner ◽  
Nicotiana Tabaccum ◽  
Calmodulin Binding ◽  
Dependent Protein

A calcium (Ca2+)/calmodulin (CaM)-binding protein kinase (CBK) from tobacco (Nicotiana tabaccum), NtCBK2, has been characterized molecularly and biochemically. NtCBK2 has all 11 conserved subdomains of the kinase-catalytic domain and a CaM-binding site as shown by other kinases, including Ca2+-dependent protein kinase and chimaeric Ca2+/CaM-dependent protein kinases. However, this kinase does not contain an EF-hand motif for Ca2+ binding, and its activity was not regulated by Ca2+. Whereas NtCBK2 phosphorylated both itself and other substrates, such as histone IIIS and syntide-2, in a Ca2+/CaM-independent manner, as also shown by OsCBK, a CaM-binding protein kinase from rice (Oryza sativa), the kinase activity of NtCBK2 was greatly stimulated by Ca2+/CaM, whereas that of OsCBK was not. By molecular dissection analyses, the CaM-binding domain of NtCBK2 has been localized in a stretch of 30 amino acid residues at residue positions 431–460 as a 1-5-10 protein motif. Three tobacco CaM isoforms (NtCaM1, NtCaM3 and NtCaM13) used in the present study have been shown to bind to NtCBK2, but with different dissociation constants (Kds), as follows: NtCaM1, 55.7 nM; NtCaM3, 25.4 nM; and NtCaM13, 19.8 nM, indicating that NtCBK2 has a higher affinity for NtCaM3 and NtCaM13 than for NtCaM1. The enzymic activity of NtCBK2 was also modulated differently by various CaM isoforms. Whereas the phosphorylation activity of NtCBK2 was shown by assay to be enhanced only ≈2–3-fold by the presence of NtCaM1, the activity could be amplified up to 8–9-fold by NtCaM3 or 10–11-fold by NtCaM13, suggesting that NtCaM3 and NtCaM13 are better activators than NtCaM1 for NtCBK2.

scholarly journals The isolation, characterization and partial sequences of the chicken erythrocyte non-histone chromosomal proteins HMG14 and HMG17. Comparison with the homologous calf thymus proteins

1980 ◽  
Vol 185 (2) ◽  
pp. 383-386 ◽  
Author(s):  
J M Walker ◽  
E W Johns
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
British Library ◽  
Chicken Erythrocyte ◽  
Terminal Sequence ◽  
Sequence Analyses ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Calf Thymus ◽  
Core Histones

Non-histone chromosomal proteins HMG14 and HMG17 were isolated from chicken erythrocyte nuclei. The proteins were characterized by amino acid analysis and by N-terminal sequence analyses. Comparison with the corresponding data for the calf thymus proteins shows that 11% of the residues in HMG14 protein and 5% of the residues in HMG17 protein differ between the two species. Proteins HMG14 and HMG17 therefore do not appear to exhibit the evolutionary stability shown by the nucleosome core histones. Detailed evidence for the amino acid sequence data has been deposited as Supplementary Publication SUP 50101 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. 4. (1978) 169, 5.

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