scholarly journals Glycosylation of ribonuclease A catalysed by rabbit liver extracts

1975 ◽  
Vol 146 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Z Khalkhall ◽  
R D Marshall
Keyword(s):  
Metal Ion ◽  
Ribonuclease A ◽  
Enzymic Activity ◽  
Rabbit Liver ◽  
Absolute Requirement ◽  
Pancreatic Ribonuclease A ◽  
Triton X ◽  
Increased Activity ◽  
Asparagine Residue

Crude extracts of rabbit liver catalyse in vitro the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to bovine pancreatic ribonuclease A. The enzymic activity is contained in rough endoplasmic reticulum. It has an absolute requirement for a bivalent metal ion: Co-2+ greater than Mn-2+ greater than Ni-2+. Mg-2+ is ineffective. There is enzymic activity in the absence of detergent, but increased activity is observed in the presence of Triton X-100. The site of glycosylation of ribonuclease A is asparagine-34, and glycosylation occurs only at this point. These findings agree with the hypothesis that the sequence Asn-X-Thr(Ser) (where X may be one of a number of types of amino acid) is a necessary, but not sufficient, condition for N-acetylglucosaminylation of a protein-bound asparagine residue.

scholarly journals Human cystathionine γ-lyase: developmental and in vitro expression of two isoforms

2000 ◽  
Vol 347 (1) ◽  
pp. 291-295 ◽  
Author(s):  
Anna-Liisa LEVONEN ◽  
Risto LAPATTO ◽  
Mika SAKSELA ◽  
Kari O. RAIVIO
Keyword(s):  
Human Liver ◽  
Human Kidney ◽  
Enzymic Activity ◽  
Developmental Changes ◽  
Transcriptional Level ◽  
Mrna Isoforms ◽  
Gestational Week ◽  
Neonatal Liver ◽  
Increased Activity

Cystathionine γ-lyase (CGL) is the last enzyme of the trans-sulphuration pathway, which converts methionine into cysteine. To study the possible differences in enzymic activity of the two human cystathionine γ-lyase isoforms characterized earlier, these were separately expressed in human kidney embryonic 293T cells. Furthermore, developmental changes in the expression of the two mRNA forms as well as the enzymic activity in human liver were studied, as it has been postulated that a change in the relative expression of CGL isoforms causes the postnatal increase in CGL activity. Transfection with the longer isoform increased the CGL activity 1.5-fold, while the activity of the cells transfected with the shorter form did not differ from the basal activity. In human liver samples, CGL activity was only detected in adult tissue (68±9 nmol of cysteine/h per mg of protein), whereas activity in fetal, premature and full-term neonatal liver tissue was undetectable. In contrast, strong mRNA expression of both mRNA isoforms was detected from the 19th gestational week onwards and the longer form of CGL appeared to be predominant. The expression of the two mRNA forms varied in parallel. In conclusion, we have shown that only cells overexpressing the longer form of CGL have increased activity, and CGL appears to be regulated at the post-transcriptional level during development.

scholarly journals Glycosylation in vitro of an asparagine sequon catalysed by preparations of yeast cell membranes

1976 ◽  
Vol 160 (1) ◽  
pp. 37-41 ◽  
Author(s):  
Z Khalkhali ◽  
R D Marshall ◽  
F Reuvers ◽  
C Habets-Willems ◽  
P Boer
Keyword(s):  
Yeast Cell ◽  
Cell Membranes ◽  
Ribonuclease A ◽  
Pancreatic Ribonuclease ◽  
Pancreatic Ribonuclease A

Preparations of yeast cell membranes can catalyse in vitro the N-acetyl-beta-D-glucosaminylation of the asparagine sequon at residues 34-36 of bovine pancreatic ribonuclease A. The relevant glycopeptides were isolated from tryptic hydrolysates of the glycosylated ribonuclease and analysed. The donor used was UDP-N-acetyl-D-glucosamine, although the mechanism of the transfer is unknown. Mn2+ ions at concentrations of 25 mM double the activity of the enzymic transfer.

scholarly journals Purification of D-alanine carboxypeptidase from Escherichia coli B on a penicillin-Sepharose column

1975 ◽  
Vol 147 (1) ◽  
pp. 131-137 ◽  
Author(s):  
M Gorecki ◽  
A Bar-Eli ◽  
Y Burstein ◽  
A Patchornik ◽  
E B Chain
Keyword(s):  
Escherichia Coli ◽  
Protein Band ◽  
Enzymic Activity ◽  
Coli Strain ◽  
Sepharose Column ◽  
Absolute Requirement ◽  
Single Protein ◽  
One Step ◽  
Triton X ◽  
Escherichia Coli B

1. A soluble D-alanine carboxypeptidase from Escherichia coli strain B was purified on a p-aminobenzylpenicillin-Sepharose column. This one-step chromatography followed by an (NH4)2SO4 precipitation yielded an enzyme purified 1200-fold and some of its properties are reported. 2. The pure D-alanine carboxypeptidase was devoid of D-alanine carboxypeptidase II activity and migrated as a single protein band on analytical disc gel electrophoresis. 3. Triton X-100 in the purification procedure is an absolute requirement for obtaining a stable enzyme. 4. The enzymic activity of D-alanine carboxypeptidase was greatly affected in solution of high salt concentrations and varied somewhat with the nature of the cation tested.

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251 ◽  
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

scholarly journals On the Preparation of Bovine Pancreatic Ribonuclease A

1963 ◽  
Vol 238 (2) ◽  
pp. 618-621 ◽  
Author(s):  
Arthur M. Crestfield ◽  
William H. Stein ◽  
Stanford Moore
Keyword(s):  
Ribonuclease A ◽  
Pancreatic Ribonuclease ◽  
Pancreatic Ribonuclease A

scholarly journals Bioactive Plasma Coatings on Orthodontic Brackets: In Vitro Metal Ion Release and Cytotoxicity

Coatings ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 857
Author(s):  
Lasni Samalka Kumarasinghe ◽  
Neethu Ninan ◽  
Panthihage Ruvini Lakshika Dabare ◽  
Alex Cavallaro ◽  
Esma J. Doğramacı ◽  
...  
Keyword(s):  
Metal Ion ◽  
Prolonged Exposure ◽  
Orthodontic Brackets ◽  
Plasma Polymer ◽  
Functional Coatings ◽  
Key Factors ◽  
Ion Release ◽  
Metal Ion Release ◽  
Surface Functionalisation

The metal ion release characteristics and biocompatibility of meta-based materials are key factors that influence their use in orthodontics. Although stainless steel-based alloys have gained much interest and use due to their mechanical properties and cost, they are prone to localised attack after prolonged exposure to the hostile oral environment. Metal ions may induce cellular toxicity at high dosages. To circumvent these issues, orthodontic brackets were coated with a functional nano-thin layer of plasma polymer and further immobilised with enantiomers of tryptophan. Analysis of the physicochemical properties confirmed the presence of functional coatings on the surface of the brackets. The quantification of metal ion release using mass spectrometry proved that plasma functionalisation could minimise metal ion release from orthodontic brackets. Furthermore, the biocompatibility of the brackets has been improved after functionalisation. These findings demonstrate that plasma polymer facilitated surface functionalisation of orthodontic brackets is a promising approach to reducing metal toxicity without impacting their bulk properties.

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