Kinds Of Proteolytic Enzymes Research Articles

Near-infrared fluorescent read-out probe for ultra-sensitive imaging of leucine aminopeptidase in vitro and in vivo

Leucine aminopeptidase (LAP), a kind of proteolytic enzyme and an indicator of many kinds of cancer. Developing effective methods for tracking endogenous LAP activity is crucial for LAP-related cancer diagnosis and treatment. In this work, we report a new near-infrared fluorescent probe: CY-P, which is capable of highly effective monitoring of endogenous LAP activity in vitro and in vivo. CY-P was constructed by incorporating a dipeptide (Cys-Leu: Cysteine-Leucine) into a water-soluble NIR-emitting fluorophore (CY–OH: a semi-cyanine scaffold). Upon exposure to LAP, the amide bond in Cys-Leu will be specifically cleaved by LAP, and subsequent an intramolecular cyclization to release the unmasked phenol-based semi-cyanine. In this process, a dramatic fluorescence enhancement at 701 nm was observed, the evaluation of LAP activity is established in terms of the relationship between fluorescence light-up efficiency and LAP activity. In our experiments, CY-P features high feedback towards LAP with various advantages, such as low detection limit (4.9 × 10−5 U mL−1), fast-response (∼9 min), good biocompatibility, and NIR emission. As expected, the probe exhibits low cytotoxicity and excellent cell membrane permeability, which is successfully used to monitor endogenous LAP activity in living cancer cells and zebrafish models. Therefore, we anticipate that this newly designed fast LAP detection platform will provide an alternative method for the studies of related diseases.

Clinical value of matrix metalloproteinase-3 in rheumatic immune disease

Rheumatic immune disease is a kind of disease caused by the abnormal of autoimmune system, the pathogenesis is complex and affected by many factors. Matrix metalloproteinases are a kind of proteolytic enzyme that can degrade extracellular matrix components, while the matrix metalloproteinase3 is the main enzyme that promote their degradation. In recent years, studies have found that matrix metalloproteinase-3 participates in the development of rheumatic immune disease. It is a potential marker not only can reflect the activity of some diseases, but also to evaluate therapeutic effect. Key words: Autoimmune diseases; Rheumatic diseases; Matrix metalloproteinase 3; Tissue inhibitor of metalloproteinases; Biomarkers

Comparative study of two proteases for mass spectrometric method for the determination HbA1C

Objective To explore the difference of two kinds of proteolytic enzymes (GLU-C and Lys-C trypsin),hemoglobin A1c were measured in mass spectrometry. Methods Based on the IFCC recommended hemoglobin A1c reference measurement method by mass spectrometry,the blood samples were preparied (the number of these blood samples:201201,201202,201203).Hemoglobin in the samples were enzymed respectively by two kinds of proteolytic enzymes (Glu-C and Lys-C),before the mass spectra which be used solid phase extraction method,under the optimal experimental conditions,hemoglobin A1c in the sample were measured by the liquid chromatography tandem mass spectrometry and screened of variant hemoglobin with sequest software.All results by mass spectrometry are required to use the Sequest software to search RAW file in the human IPI Database,retrieve four common hemoglobin variant published results and compare hemoglobin variant sites,using statistical software SPSS13.0 and Excel 2003 on the results in the correlation analysis,compare between groups by the independent sample t test. Results In the detection of hemoglobin A1c,respectively Glu-C and Lys-C blood which the samples were enzymed,results of hemoglobin A1c by Lys-C were (34.70±2.80),(51.76±1.60),(73.39±1.11) mmol/mol,results of hemoglobin A1c by Glu-C were (33.12±1.48),(54.54±2.50),(75.40±3.60) mmol/mol,the difference between groups was not statistically significant,the results of two methods of drawing curve,all R2>0.995.Searching the human database with sequest software,four common hemoglobin variant were not be finded. Conclusions In the detection of hemoglobin A1c by the mass spectrometry,the application of the above two kinds of proteolytic enzyme in the blood samples,that can be more consistent results,test results of two methods have a good correlation.(Chin J Lab Med,2013,36:733-737) Key words: Hemoglobin A; glycosylated; Hemoglobins; Serine endopeptidases; Trypsin; Chromatography; high pressure liquid; Tandem mass spectrometry

Analysis of Structures, Functions, and Epitopes of Cysteine Protease fromSpirometra erinaceieuropaeiSpargana

Spirometra erinaceieuropaei cysteine protease (SeCP) in sparganum ES proteins recognized by early infection sera was identified by MALDI-TOF/TOF-MS. The aim of this study was to predict the structures and functions of SeCP protein by using the full length cDNA sequence of SeCP gene with online sites and software programs. The SeCP gene sequence was of 1 053 bp length with a 1011 bp biggest ORF encoding 336-amino acid protein with a complete cathepsin propeptide inhibitor domain and a peptidase C1A conserved domain. The predicted molecular weight and isoelectric point of SeCP were 37.87 kDa and 6.47, respectively. The SeCP has a signal peptide site and no transmembrane domain, located outside the membrane. The secondary structure of SeCP contained 8 α-helixes, 7 β-strands, and 20 coils. The SeCP had 15 potential antigenic epitopes and 19 HLA-I restricted epitopes. Based on the phylogenetic analysis of SeCP, S. erinaceieuropaei has the closest evolutionary status with S. mansonoides. SeCP was a kind of proteolytic enzyme with a variety of biological functions and its antigenic epitopes could provide important insights on the diagnostic antigens and target molecular of antisparganum drugs.

Cartilage Destruction Is Partly Induced by the Internal Proteolytic Enzymes and Apoptotic Phenomenon of Chondrocytes in Relapsing Polychondritis

We analyzed 9 cases by immunohistochemical studies in order to elucidate the mechanisms of cartilage destruction in relapsing polychondritis (RP), which often involves the external auricle and respiratory tract through immunological disorder. Cartilage tissues were obtained during surgical operations. Cell species in the granulation tissues, especially near the cartilage, were identified by cell-surface markers [CD3, CD4, CD8, CD20, CD45 (LCA), and CD68]. The proteolytic enzymes expressed in the cells in the perichondral granulation and in chondrocytes themselves were analyzed by immunohistochemical studies using anti-matrix metalloproteinase (MMP) -1, -3, -8, -9, and -13, and cathepsin D, K, L, and elastase antibodies. Apoptosis and nitric oxide (NO), an apoptosis-related factor, were also examined using ApopTag and antinitrotyrosine antibody, respectively. Among cell species that infiltrated in perichondral granulation, LCA, CD68 (monocytes/macrophages), and CD4 cells were dominant in number; MMP-8, MMP-9, and elastase were expressed only in the perichondral granulation; whereas MMP-3 and cathepsin K and L were detected in both chondrocytes and granulations. Out of 9 cases examined, 6 revealed apoptotic cells in excess of 50% of chondrocytes. There was a strong correlation between the number of apoptotic cells and the number of MMP-3-positive (r = 0.83) and cathepsin K-positive cells (r = 0.92). Abundant NO-expressing cells were observed in the chondrocytes in degenerated cartilage, similar to apoptosis. Cartilage destruction in polychondritis is induced not only by perichondral inflammation, but also by intrinsic factors expressed in chondrocytes themselves, including certain kinds of proteolytic enzymes and apoptosis.

물리적 처리에 따른 우육추출물중의 BSA와 BGG단백질의 SDS-PAGE패턴 및 항원성의 변화

이전의 연구에서, 우육중의 주된 알레르겐은 우육추출물 중의 BSA, BGG라고 보고하였다. 본 연구에서는 이들 단백질의 열처리와 초고압처리와 같은 물리적 처리에 따른 SDS-PAGE상의 변화와 우육알레르기 환자의 혈청을 이용한 알레르겐성의 변화를 Western blots로 관찰하였다. 우육 추출물에 대한 열처리에 의한 알레르겐 단백질의 변화에서, 100℃ 가열처리군에서 BSA band는 큰 감소를 보였으나, BGG band는 완전히 제거되지 못하고 남아 있었고, 항원성 역시 대조군에 비하여 감소는 하였으나 여전히 유지되고 있었다. 120℃이상의 열처리는 추출물중의 BSA와 BGG band의 소실과 이에 따른 항원성의 큰 감소를 가져왔다. 우육추출물중의 BSA단백질은 열처리에서와 달리 초고압처리에 의하여 전기영동상에 거의 변화를 보이지 않았다. 그러나 BGG 단백질은 400 M㎩ 가압처리군부터 전기영동상 band가 감소하였고, 항원성 역시 유의적으로 감소하였다. 이는 초고압처리 정도에 따른 우육추출물중의 BSA와 BGG를 포함한 여러 분자의 SDS-PAGE상의 특이적 변화가 최초로 시사된 결과이다. 이러한 BGG 단백질의 고압처리에 의한 변화는 추출물중의 어떤 효소에 의한 것으로 추측되었고, 고압처리 후 37℃숙성 실험으로 이러한 가설이 확인되었다. 우육추출물에 존재하고 초고압처리로 활성화되어 BGG분자에 특이적으로 관여하는 효소 및 이와 관련된 메커니즘에 대한 연구가 앞으로 필요할 것으로 보인다.

Biodegradation of ornithine-containing polylysine hydrogels

The degradation of the cross-linked cationic poly(amino acid)-glutaraldehyde (GA) hydrogels by two kinds of proteolytic enzymes, trypsin and Aspergillus Protease Type XXIII, and by seven species of soil filamentous fungi has been investigated using homo- and copolypeptides of lysine (Lys) and ornithine (Orn). Trypsin degraded the hydrogels prepared from poly(Lys) and copoly(Lys Orn)s but not poly(Orn), while Aspergillus protease degraded all of them. Degradation time of hydrogels by the two proteases became longer with increasing Orn content in the gel. Seven species of soil filamentous fungi were cultured with hydrogels on Czapeck medium to evaluate the degree of microbial degradation of the hydrogels, and the three species of the fungi, Aspergillus oryzae, Penicillium citrinum and Curvularia sp., were grown in culture with an accompanying degradation of the gel matrix, while the other four species, Mucor sp., Rhizopus sp., Cladosporium sp., and Trichoderma sp., were not. The degree of degradation of gel matrix with growth of the three fungi became lower with increasing Orn content in the gel matrix. The results might offer some clues to the applications for the controlled biodegradation of cationic poly(amino acid) hydrogel by introduction of Orn, suggesting that unnatural amino acid resists hydrolysis by proteases or microorganisms.

Properties of staphylococcal enterotoxin a under limited protolysis

1. 1. Staphylococcal enterotoxin A (SEA) was exposed in a state of limited proteolysis to five kinds of proteolytic enzymes: papain, pepsin, pronase, trypsin and α-chymotrypsin. SEA was found to be sensitive to the action of three of them: papain, pepsin and pronase. 2. 2. Four fragments were produced after papain proteolysis of SEA in the presence of β-mercaptoethanol. 3. 3. Papain processing of SEA does not influence its capacity to interact with antibodies to intact toxin, but the capacity of SEA to hydrolyse NAD to nicotinamide (NA) and adenosinediphosphatribose (ADP-ribose) completely disappears. 4. 4. SEA under the action of pepsin and pronase has been hydrolysed to small peptides, which appear to be moving with the front of leading dye in disc-electrophoresis.

Preliminary crystallographic data for an extracellular proteolytic enzyme from a strain of Arthrobacter

The chapter discusses endopeptidases and focuses on their three main types: (1) acid proteases, (2) diisopropylphosphofluoridate (DFP)-sensitive alkaline proteases, and (3) metal chelator-sensitive neutral proteases. The acid proteases that include the Aspergillus saitoi acid protease, Rhizopus chinensis acid protease, and Paecilomyces varioti acid protease are characterized by low pH activity and stability profiles, insensitivity to metal chelating agents, phosphorylating compounds, such as DFP and thiol poisons. These enzymes show limited esterolytic activity and are reminiscent of the animal pepsins and rennins. The most widely distributed group of proteolytic enzymes of both microbial and animal origin is that of the the serine proteases, generally identified by their sensitivity to organophosphorus reagents, such as DFP and isopropylmethylphosphofluoridate. In addition to the three broad classes of endopeptidases, a number of different kinds of proteolytic enzymes are produced by bacteria and molds. Another unique group of proteases, which in some cases shares properties with one of the other groups, is the bacterial cell wall lysing proteases. These enzymes by virtue of the specific bonds cleaved have uniquely difierent characteristics from the other enzymes.

TREATMENT OF CHRONIC SINUSITIS MAXILLARIS WITH PRONASE-P Part 3

Biochemical studies were made in Pronase-Porally administered cases of chronic sinusitis maxillaris to clarify the drug's healing mechanism which has been only partially reported. Of the various kinds of proteolytic enzymes those with powerful hydrolysis seem favorable for a pus-dissolving action in clinical use. In this report, Pronase-P is found to be most satisfactory, followed by a-chymotripsin and Varidase in descending order.In cases where responses to a single oral administration of Pronase-P were not seen, favorabe results were obtained with a combined administration of antibiotics.

Properties of Resilin, a Rubberlike Protein

Abstract There is much circumstantial evidence in favor of the belief that elastin and some other structural proteins exist in the rubberlike state of matter (see reviews by Wöhlisch, 1939; Astbury, 1940; Meyer, 1950; Kendrew, 1954), but it will be shown that the crucial evidence from thermodynamic experiments is ambiguous. The discovery of a new type of highly elastic structural protein, resilin ( Weis-Fogh, 1960; Bailey and Weis-Fogh, 1961), made it possible to re-investigate the problem by using more suitable samples. Since the thermodynamic experiments reported here gave a clear-cut answer, it was legitimate to interpret in detail the mechanical and optical properties of resilin according to the kinetic theories of rubber elasticity and so to obtain valuable information about the molecular structure of at least one rubberlike protein (Weis-Fogh, in preparation). Resilin is the essential elastic component in certain mechanical springs in the cuticle of arthropods (cf. the Latin resilire, to spring back) and it has a number of properties which make it ideally suited for this type of investigation (Weis-Fogh, 1959; 1960). When swollen with water, it exhibits typical long-range elasticity, it is heat-stable up to 140° C, crosslinked by very stable covalent bonds and insoluble in all solvents that do not break the peptide backbone but it is easily digested by all kinds of proteolytic enzymes. Moreover, it is secreted as thick continuous layers by the epidermis in a pure form unmixed with other proteins, lipids or polysaccharides and spatially separated from chitin, the only other structural component of the rubberlike cuticle (Weis-Fogh, 1960; Bailey and Weis-Fogh, 1961). It is thus possible to obtain native resilin in the form of relatively large, homogeneous samples, 0.1 mm thick and 1 mm long. Such samples are hyaline, devoid of color, and mechanically as well as optically isotropic in all directions and at all degrees of swelling. They are also devoid of structure (light and electron microscopy), and no trace of crystallinity has been found (X-ray diffraction) even in samples that were stretched near to the breaking point and then slowly dried (Elliott, Huxley, and Weis-Fogh, in preparation). The amino acid composition is unique and different from that of elastin, elastoidin, silk fibroin and collagen, and about one third of the side chains carry polar groups (Bailey and Weis-Fogh, 1961). The rubberiness of elastin is generally thought to be intimately connected with the scarcity of polar groups (5%; cf. Lloyd and Garrod, 1946). This paper will deal with experiments that show that the elastic force of resilin is connected with entropy changes caused by deformation, as in true rubbers, rather than with changes in internal energy, as in most solids.