Functional Activity Of Protein Research Articles

EFFECTS OF INFRARED LASER RADIATION OF LOW INTENSITY ON HEMOSTASIS SYSTEM

Roentgenostructural, spectral analyses, functional testing of the hemostatic system in experimental animals demonstrated that infrared low-intensity laser radiation changes functional activity of proteins of the hemostatic system. The effect of the radiation depends on the dose and frequency of the radiation impulses. One of the complications is DIC-syndrome which may develop if the exposure dose on the radiation point in tissues with blocked circulation reaches 9.5 mJ/cm 2 and higher.

Antibodies directed to protein S in patients with systemic lupus erythematosus: prevalence and clinical significance.

Antibodies directed against protein S (anti-ProtS) may be involved in the development of thrombosis in patients with the antiphospholipid syndrome. We assessed the prevalence and clinical significance of anti-ProtS and evaluated their immunological characteristics in 184 patients with SLE and 99 healthy donors. All patients were tested for IgG anti-ProtS by an in-house ELISA. Plasma levels and functional activity of protein S were also tested. Anti-ProtS were found in 57 patients (31%) and 4 healthy controls (4%). Patients with thrombosis had anti-ProtS more frequently than controls (29% vs 4%, OR 9.5 [95% CI 3.07-29.3], p<0.0001). Anti-ProtS were more frequent in patients with venous thrombosis and in those with arterial thrombosis, than in controls (41% vs. 4%, OR 16.5 [95% CI 5-54], p<0.0001 and 23% vs. 4%, OR 7 [95%CI 2.1-23.5], p=0.0008, respectively). Patients with prematurity, preeclampsia and intrauterine growth restriction had anti-ProtS more frequently than the control group (36%, 47% and 44% vs. 4%; OR 13.6 [95% CI 2.8-66], p=0.003, OR 21 [95% CI 5-86], p<0.0001 and OR 19 [95% CI 4-99], p=0.0014, respectively). Plasma levels of free protein S were not statistically different between patients with and without anti-ProtS and controls (77.9% [20.7-100] vs. 83.7% [52.7-100] vs. 89% [62-101], respectively). Free protein S functional activity was no different between subgroups (105% [48-230] in anti-ProtS positive vs. 123% [95-283] in anti-ProtS negative vs. 136% [60-174] in controls). Anti-ProtS are frequent in SLE patients with thrombosis and pregnancy morbidity. These antibodies do not interfere with free protein S in plasma since its level and/or functional activity are not impaired.

タンパク質結晶学のブレイクスルー １． タンパク質結晶構造解析法の進展 コムギはい芽を用いた無細胞タンパク質合成法

With the sequencing of the genomes of various species, attention has turned to the structure, properties, and functional activities of proteins. However, rapid progress in the area of proteomics is premised on the availability of sufficient amounts of a large number of proteins. Here we described a novel cell-free system from wheat embryos for the high-throughput screening/ synthesis of gene products. Our system should open up many possibilities in the post-genome era.

Production of Antibodies That Recognize Specific Tyrosine‐Phosphorylated Peptides

Phosphorylation of tyrosine residues of various proteins plays an important role in regulation of the functional activities of proteins within the cell. Antibodies to specific tyrosine-phosphorylated peptides are a valuable tool for analysis of the localization and function of individual phosphoproteins. This unit describes the methods used to produce and screen monoclonal and polyclonal antibodies for specificity for only the phosphorylated state of a peptide.

Expression of rat, renal NHE2 and NHE3 during postnatal developmental

The current studies were designed to characterize the expression of sodium–hydrogen exchangers NHE2 and NHE3 during rat, renal ontogeny. NHE2 mRNA and immunoreactive protein were more highly expressed at 2 and 3 weeks of age, with declining levels into adulthood. In situ hybridization of NHE2 mRNA localized the message to the renal inner cortex and outer medullary regions and suggested higher mRNA levels in suckling animals as compared to adults. Immunohistochemical analysis of rat kidney with the NHE2 antiserum showed specific staining of the distal convoluted tubules. In contrast, NHE3 mRNA expression was lowest in 2-week animals and higher in older rats, while NHE3 immunoreactive protein showed constant expression levels during development. Additionally uptake experiments utilizing HOE694 showed no change in NHE2 or NHE3 functional protein expression in 2-week-old rats versus adults. We conclude that the developmental increase in NHE2 mRNA and immunoreactive protein expression cannot be detected by functional assays, which suggests that NHE2 does not play a role in sodium absorption by the renal tubules (as has been previously suggested). Additionally, molecular changes seen in NHE3 mRNA expression do not affect functional protein activity, suggesting increased mRNA translational efficiency or protein stability in suckling rats.

Two-component signal transduction in Bacillus subtilis: how one organism sees its world.

Variability and adaptability are crucial characteristics of organisms possessing the ability to survive and prosper in a wide variety of environmental conditions. The most adaptable bacteria contain a large reservoir of genetic information encoding biochemical pathways designed to cope with a variety of environmental situations. Organisms that have the genetic capability to respond to altered conditions do so when stimulated by specific signals. Recognition of specific signals and conversion of this information into specific transcriptional or behavioral responses is the essence of signal transduction. A mechanism commonly found in bacteria for signal transduction is the two-component system (23, 26). Its basis is the conversion of signal recognition to a chemical entity, i.e., a phosphoryl group, that modifies the functional activity of proteins. Signal recognition and transduction are the province of the sensor histidine kinase component of the system. This protein has separable sensor and histidine phosphotransferase domains that function to recognize (bind) the signal, causing the kinase to autophosphorylate a histidine residue of the phosphotransferase domain (Fig. ​(Fig.1).1). The phosphoryl group is subsequently transferred to the second component protein, the response regulator, where it resides as an acyl phosphate of an aspartic acid residue. The response regulator consists of the phosphorylatable aspartate domain and an output domain that is activated to carry out its function by conformational or, perhaps, electrostatic alterations induced by the phosphoryl group. In most cases, the response regulator is a transcription activator for genes whose products are specifically utilized to respond to the unique nature of a given input signal. In the chemotaxis system of bacteria, the response regulator determines the direction of rotation of the flagellar motor. The basics of the signal transduction mechanism remain the same regardless of the input signal or the function of the response regulator. FIG. 1 Schematic view of two-component and phosphorelay systems. Activation signals recognized by sensor domains of histidine kinases result in autophosphorylation of a histidine in the histidine phosphotransferase domain (His PTase). The phosphoryl group (P) ... This phosphoryl group-based signal transduction mechanism exists in two major conformations in microorganisms: the two-component system and a four-component system termed the phosphorelay (Fig. ​(Fig.1).1). Signal interpretation and transduction by histidine kinases are the same in both, but the target of the kinase in a phosphorelay is a single-domain response regulator consisting of only the phosphorylated aspartate domain. This phosphorylated protein serves as a substrate for a phosphotransferase that transfers the phosphoryl group to a response regulator-transcription factor. The phosphotransferase is transiently phosphorylated on a histidine during this process. In a phosphorelay, the phosphoryl group is transferred in the order His-Asp-His-Asp, which differs from the His-Asp series of a two-component system. In the first-discovered phosphorelay used to initiate sporulation in Bacillus subtilis, all of the components (domains) reside on different proteins (4). Subsequently discovered phosphorelays in bacteria, fungi, and plants use composite proteins where the kinase and first response regulator domain and sometimes the phosphotransferase domain are contiguous within a single polypeptide chain (1, 6). The sporulation initiation phosphorelay is a signal integration circuit that processes both positive and negative signals, which suggests that phosphorelays are used where a number of opposing signals must be interpreted by the signal transduction system (22).

The PKR protein kinase--an interferon-inducible regulator of cell growth and differentiation.

Post-translational modifications such as protein phosphorylation provide an important mechanism by which the functional activity of proteins can be controlled and, hence, biological processes regulated. Interferons (IFN) are a multigene family of cytokines that can profoundly affect a wide variety of functions in animal cells including virus replication, cell growth and differentiation, and the immune response. Changes in protein phosphorylation mediated by the IFN-inducible, RNA-dependent protein kinase (PKR) are implicated in the control of cell proliferation mediated by IFNs. Our knowledge of the structure, regulation and function of PKR will be summarized in this brief review, with focus on those aspects of protein phosphorylation and interferon action involving PKR that are central to the roles of the enzyme in the control of cell growth and proliferation.

A Functional Protein S and Microlatex Immunoassay for Protein S and C4b-Binding Protein on the Automated Coagulation Laboratory (ACL) 300 Plus

Protein S can be determined by functional or immunological assays. Electroimmunodiffusion (EID) or enzyme immunoassays (enzyme-linked immunosorbent assay; ELISA) are the commonly employed techniques for measuring protein S and C4b-binding protein (C4b- BP) immunologically. Procedures for these assays are time-consuming and labor-intensive. The introduction of microlatex immunoassays (LIATEST system; Diagnos tica Stago, Asnieres-Sur-Seine, France) has provided an alternative for rapid and reliable immunological determi nation. We have placed the microlatex immunoassay for total and free protein S (TPS, FPS) and C4b-BP, using the light-scattering mode, on the Automated Coagulation Laboratory (ACL) 300 Plus (Instrumentation Laboratory, Lexington, MA, U.S.A.). We also placed a functional activity assay of protein S (STACLOT protein S; Amer ican Bioproducts, Parsippany, NJ, U.S.A.) on the ACL 300 Plus. The performance characteristics for the assays yielded a within-run coefficient of variance (CV) of 2.5- 4.6% ( n = 13) for TPS, 4.0-4.8% ( n = 13) for FPS, 1.9- 3.0% ( n = 11) for C4b-BP, and 2.3-5.9% for protein S activity. The interrun CV was 2.1-5.7% ( n = 24), 3.7- 7.0% ( n = 12), 2.6-7.0% ( n = 16), and 4.0-8.4% ( n = 27), respectively. Analytical recovery was 94-109, 97-100, 91-103, and 99-103%, respectively. The normal ranges determined on plasmas from 30 healthy individuals were 113 ± 37 (mean ± 2 SD) for TPS, 106 ± 35 for FSP, 111 ± 22 for C4b-BP, and 107 ± 34 for protein S activity. The results for the microlatex immunoassay and either the EID or the ELISA methods showed excellent correla tions for FPS and C4b-BP; the correlations between LIATEST and either EID or ELISA for TPS were also relatively high. The functional activity of protein S cor related well with FPS. Microlatex immunoassays, using the light-scattering mode for TPS, FPS, or C4b-BP, and the functional assay of protein S can be adapted on the ACL 300 Plus system with a high accuracy and reproduc ibility and with considerable time saving.

Effect of urea on the structure and functional activity of proteins. Chymotrypsinogen A and α-chymotrypsin

The effect of urea on the structural stability and functional activity of globular proteins,viz., chymotrypsinogen A (ChtG) and α-chymotrypsin (Cht), was studied over a wide range of concentrations (0.5–6 rnol L-1), and the existence of two different mechanisms of the action of urea on these proteins was demonstrated. No changes in the spatial structure of ChtG were observed in the concentration range from 0,5 to 3 mol L−1 (region 1). Differential UV spectroscopy shows tile redistribution of aromatic groups between the inner volume and the outer surface of a protein molecule (protein denaturation) at concentrations >3 mol L−1 (regionII), In regionI, urea changes the kinetic parameters of enzymatic reactions involving Cht, which is explained on the basis of millimeter spectroscopy data by its action on the structure and nucleophilic reactivity of water.

Laboratory testing for hypercoagulable disorders

Evaluation of inherited hypercoagulability in patients with venous thromboembolism includes testing for the functional activity of protein S, protein C, antithrombin III, and for resistance to activated protein C. Resistance to activated protein C can be assessed with plasma as well as with DNA-based assays that are commercially available. Acquired disorders include the development of antibodies against phospholipid-protein complexes (as would occur in patients with the antiphospholipid syndrome). The inherited and acquired abnormalities are most commonly apparent as superficial or deep venous thromboembolism in younger patients, most of whom have additional risk factors for thrombosis, such as use of oral contraceptives or recent trauma. These tests are cost effective if the results will influence patient management or will have potential value in the care of family members in situations of increased thrombotic risk.

Functional Activity of Protein C in Newborn Infants

Protein C is lower in newborn infants than in adults. There are conflicting reports regarding functional activity and the presence of des-gamma carboxylated species in the newborn. We have compared protein C activity and antigenic level in newborn infants and found the activity: antigen ratio to be lower than in adults (0.69 versus 1.0). We discuss this finding in relation to the previously published reports.

Characterization of protein G expressed by human group C and G streptococci

Treatment of human group C and G streptococci with cyanogen bromide results in solubilization of surface protein G molecules. Strain to strain variation in the quantity, molecular weight, and functional activity of protein G extracted from representative group C and G isolates led to the identification of five structurally and functionally distinct forms of the protein. These forms included molecules containing: (i) 3 albumin and 3 igG binding domains; (ii) 3 albumin and 2 IgG binding domains; (iii) 2 albumin and 2 IgG binding domains; (iv) 0 albumin and 3 IgG binding domains; and (v) 0 albumin and 2 IgG binding domains. Only a single form of protein G was expressed by each streptococcal isolate and this was found to be a stable characteristic for that strain.

Functional activity of protein S determined with use of protein C activated by venom activator.

A new screening procedure, an easy and specific assay for determining functional Protein S activity, has been developed, with use of Protein C activated by venom activator (Protac). Purified Protein C (100% amidolytic activity) was activated by venom activator (6 units/mL). To a mixture of 100 microL of Protein S-deficient plasma, 20 microL of sample plasma, 100 microL of cephalin (Actin), and 20 microL of activated Protein C we added 100 microL of 25 mmol/L CaCl2 solution and measured the clotting time with a KC 10 coagulometer. The functional Protein S activity correlated well with the concentrations of Protein S antigen measured by enzyme immunoassay and the Laurell rocket technique (r = 0.810 and 0.850, respectively) in normal subjects, patients with myocardial infarction undergoing warfarin therapy, and patients with liver cirrhosis.

Changes in antioxidative activity and composition of blood erythrocyte phospholipids induced in neurotic patients by phenazepam

Exposure of experimental animals and man to various stressor influences gives rise to intensification of free-radical lipid peroxidation (LPO) of cell membranes [4, 9]. The intensity of LPO is interconnected with the physicochemical properties of membrane lipids [3] and with functional activity of proteins, including enzymes, receptors, and channelforming units, dependent O n them. Considering the importance of emotional stress in the genesis and development of neurotic disorders, it can be tentatively suggested that a definite role in their pathogenesis is played by changes in peroxidation processes.

Plasma protein s activity measured using protac, a snake venom derived activator of protein C

Functional activity of protein S, a cofactor of activated protein C-dependent inhibition of blood coagulation, in human plasma was measured by using Protac, a snake venom derived activator of protein C. This assay appeared to be specific for protein S, because 1) the activated partial thromboplastin time of protein S-depleted plasma depended on the purified protein S added in the presence of Protac; and 2) the level of protein C in plasma sample (0 to 10 μg/ml) had no influence on the clotting time. The cofactor activity of protein S in the plasma of normal men (n=16) and women (n=14) was 99.4 ± 23.8% and 98.6 ± 24.5% respectively. The protein S activity in the plasma of pregnant women at pre- and post-partum (n=14), and that in the plasma of patients under warfarin therapy (n=20) were 46.2 ± 18.9%, 45.8 ± 19.6% and 24.0 ± 15.7%, respectively. In these plasmas, the levels of protein S activity were lower than those of total protein S antigen, but were similar to those of free protein S antigen. In 16 patients out of two families with congenital protein S deficiency, the protein S activity, the free antigen and the total antigen were 9.4 ± 6.9%, 13.3 ± 4.6% and 57.4 ± 20.7%, respectively. There was no significant relationship between the level of protein S activity and that of a complemental C4b-binding protein antigen in any of these patients.

Effect of adenosine on concanavalin A agglutination of human erythrocytes

We have attempted to correlate the functional activity of protein 3 with its activity as a receptor for concanavalin A. The concanavalin A agglutination of human erythrocytes is enhanced by adenosine. It varies with time of storage of the blood and is dependent on the concentration of adenosine in the medium. Adenine and/or inosine, which increase cellular ATP, do not substitute for adenosine in enhancing agglutination, and adenosine enhances agglutination of fresh erythrocytes with normal levels of ATP. Thus, it appears that cellular ATP levels are not directly involved in modulation of concanavalin A agglutination by adenosine. Trypsin, which hydrolyzes most of the exposed proteins of the cell surface but does not alter protein 3, enhances concanavalin A agglutination without altering the relative response of the cell to adenosine. Glucose, as well as the glucose transport inhibitors maltose and cellobiose, inhibits agglutination. High concentrations of adenosine reverse the inhibition by glucose and enhance agglutination in the presence of maltose and cellobiose. Treatment of erythrocytes with 4,4′-diisothiocyanostilbene-2,2-disulfonic acid disodium salt, which selectively inhibits the anion transport function of protein 3, substantially inhibits adenosine-supported concanavalin A agglutination. Treatment of erythrocytes with iodoacetate under conditions in which it selectively reacts with glyceraldehyde-3-phosphate dehydrogenase inhibits agglutination. Adenosine protects this dehydrogenase in erythrocytes from inactivation by iodoacetate, over the same concentration range in which it enhances agglutination.