Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.
Aspartic Acid/chemistry* ; Bacterial Outer Membrane Proteins/metabolism ; Blotting, Western ; Escherichia coli/enzymology* ; Escherichia coli/chemistry ; Micrococcal Nuclease/metabolism ; Mutagenesis, Site-Directed ; Oligonucleotides ; Protein Precursors/metabolism ; Serine Endopeptidases/metabolism* ; Serine Endopeptidases/genetics ; Serine Endopeptidases/chemistry* ; Structure-Activity Relationship

Animals ; Hepatocyte Growth Factor ; metabolism ; Humans ; Proto-Oncogene Proteins c-met ; Serine Endopeptidases

OBJECTIVE: To investigate the changes of the activity of tryptase of sera, lungs and bronchial tubes in the guinea-pigs which suffered from hetero-serum anaphylactic shock. METHODS: Sera and tissues were collected from anaphylactic shock guinea-pigs, and the enzyme activity was tested colormetrically using special substrate, BAPNA. RESULTS: The activity of tryptase of sera, lungs and bronchial tubes increased significantly in Anaphylactic guinea-pigs compared with control group. CONCLUSION The changes of tryptase activity are helpful to diagnose anaphylactic shock.
Anaphylaxis/enzymology* ; Animals ; Female ; Forensic Medicine ; Guinea Pigs ; Male ; Serine Endopeptidases/metabolism* ; Tryptases

OBJECTIVETo investigate the time course of changes in the expression of fibroblast activation protein (FAP) during healing of skin scald burns in rats.

METHODSAdult Wistar rats were randomized into two equal groups (n=42) and subject to superficial second degree and deep second degree scald burns on the dorsal skin groups, with 6 normal rats serving as the control group. At 6 h, 12 h, and 1, 3, 7, 14, and 21 days after burns, 6 rats from each group were sacrificed to detect FAP expression by immunohistochemistry and Western blotting.

RESULTSFAP was expressed on the cell membrane and in the cytoplasm of the fibroblasts, especially those around the neovessels. In both burn groups, FAP expression increased significantly at 6 h after burns. In superficial burn group, FAP expression was comparable between 6 and 12 h and between 1 and 3 days (P>0.05), but showed significant differences between the other time points (P<0.05). In deep burn group, FAP expression was comparable between 12 h, 1 day and 3 days (P>0.05) but differed significantly between the other time points (P<0.05). In both burn groups, FAP expression reached the peak level at 7 days followed by a gradual declination. At 21 days after the burns, FAP maintained a significantly higher expression level than the control level (P<0.05).

CONCLUSIONThe time course of the changes of FAP expression following scald burns suggests an important role of FAP in the healing process of scald burns.

Animals ; Burns ; metabolism ; rehabilitation ; Face ; Gelatinases ; metabolism ; Membrane Proteins ; metabolism ; Rats ; Rats, Wistar ; Serine Endopeptidases ; metabolism ; Skin ; metabolism ; Wound Healing

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) all over the world, there is an increasing number of children with such infection. Angiotensin-converting enzyme 2 (ACE2), one of the binding sites for SARS-CoV-2 infection in humans, can bind to viral spike proteins, allowing transmembrane serine protease (TMPRSS2) to activate S-protein to trigger infection and induce the production of various inflammatory factors such as interleukin-1, interferon-l, and tumor necrosis factor. Compared with adults, children tend to have lower expression levels of ACE2 and TMPRSS2, which are presumed to be associated with milder symptoms and fewer cases in children. The article summarizes the research advances in the role of ACE2 during SARS-CoV-2 infection, in order to help understand the pathogenic mechanism of SARS-CoV-2 and provide a reference for better development of drugs and vaccines to prevent and treat coronavirus disease 2019 in children.
Angiotensin-Converting Enzyme 2/metabolism* ; COVID-19 ; Child ; Humans ; Receptors, Virus/metabolism* ; SARS-CoV-2 ; Serine Endopeptidases/metabolism*

In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.
Bacillus ; genetics ; metabolism ; Enzyme Stability ; Fermentation ; Genetic Engineering ; Metals ; pharmacology ; Recombinant Proteins ; biosynthesis ; Serine Endopeptidases ; genetics ; isolation & purification ; metabolism

Limulus Factor C, a serine protease zymogen from the amoebocytes of the limulus, has high affinity for endotoxin. When Factor C is activated by endotoxin, it hydrolyses artificial tripeptide substrate and measurable products are released, so it can be used as an alternative reagent for endotoxin analysis. Factor C gene of Tachypleus tridentatus was obtained through RT-PCR and the recombinant protein was expressed by Bac-to-Bac/BmNPV baculovirus expression system in silkworm larvae. The activity of Factor C was detected with diluted serum of silkworm larvae, and the sensitivity of endotoxin detected was 0.2 EU/mL when the serum was diluted at 1:500. The silkworm larvae expressed limulus Factor C could be used to develop a new low-cost endotoxin test reagent.
Animals ; Arthropod Proteins ; biosynthesis ; Baculoviridae ; Bombyx ; metabolism ; Enzyme Precursors ; biosynthesis ; Genetic Vectors ; Larva ; metabolism ; Recombinant Proteins ; biosynthesis ; Serine Endopeptidases ; biosynthesis

Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.
Agkistrodon ; Animals ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics ; Thrombin ; biosynthesis ; genetics ; Viper Venoms ; enzymology

Assisted hatching (AH), which is known to improve the hatching potential of mammalian embryos, has been used to increase the pregnancy rate in in vitro fertilization cycles. However, the effect of AH on a trypsin-like protease, which is known to be associated with the hatching process, has not been studied. In this study, we evaluate whether the intactness of zona pellucida affects the secretion of a trypsin-like protease from mouse blastocyst. Four- to 8-cell stage mouse embryos were collected at 66- to 68 hr after hCG injection and divided into 3 groups according to the manipulation of zona pellucida. The groups are no treatment (control), drilling of zona pellucida (ZD) and thinning of zona pellucida (ZT). The activity of a trypsin-like protease, blastocyst development and hatching rate were compared among the three groups at 110 and 135 hr after hCG injection, respectively. The protease activity and blastocyst development were not significantly different among control, ZD and ZT groups at 110 and 135 hr after hCG injection, respectively. However, the hatching rate of ZD and ZT groups was significantly higher than that of control group at each time, respectively (p>0.001). Even in the zona pellucida removed embryos, the protease activity did not differ from the control group. In conclusion, the secretion of a trypsin-like protease from mouse blastocyst does not seem to be affected by the intactness of zona pellucida.
Animal ; Blastocyst/secretion ; Blastocyst/enzymology* ; Female ; Fertilization in Vitro/methods ; Gonadotropins, Chorionic/pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred CBA ; Pregnancy ; Serine Endopeptidases/secretion ; Serine Endopeptidases/metabolism* ; Zona Pellucida/physiology* ; Zona Pellucida/drug effects

UNLABELLED: OBJECTIVE To investigate the time-dependent expression of fibroblast activation protein (FAP) and alpha-smooth muscle actin(alpha-SMA) during the incised wound healing of the skin in mice. METHODS: The expression of FAP and alpha-SMA in incised wound of mice skin was detected by immunohistochemistry and Western blot. RESULTS: By immunohistochemistry, the expression of FAP and alpha-SMA in the normal skin and the skin 1 h after injury maintained at a very low level, but the positive cells expressing FAP and alpha-SMA started to elevate 6 h after injury and reached its peak on 5 d for FAP and on 3 d for alpha-SMA, then gradually decreased to the normal level on 14 d. The expression of FAP and alpha-SMA was observed throughout the wound healing stages 1 d after injuries by Western blot as well with a peak expression occurring on 5 d for FAP and on 3 d for alpha-SMA after injury. CONCLUSION FAP may be a potentially useful marker for wound age determination and alpha-SMA may be used as an effective indicator for the mid- and late stage incised wound of mice skin. The combination use of FAP and alpha-SMA may be potentially effective indicators for wound age determination.
Actins/metabolism* ; Animals ; Blotting, Western ; Disease Models, Animal ; Endopeptidases ; Female ; Fibroblasts/metabolism* ; Forensic Pathology ; Gelatinases/metabolism* ; Immunohistochemistry ; Male ; Membrane Proteins/metabolism* ; Mice ; Random Allocation ; Serine Endopeptidases/metabolism* ; Skin/metabolism* ; Time Factors ; Wound Healing ; Wounds and Injuries/physiopathology*