scholarly journals Preparation and properties of amphipathic enzyme-polymer conjugates

1979 ◽  
Vol 181 (1) ◽  
pp. 111-118 ◽  
Author(s):  
R A G Smith
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Ionic Strength ◽  
High Molecular Weight ◽  
Immiscible Liquid ◽  
High Ionic Strength ◽  
Polymer Conjugates ◽  
Ionic Detergents ◽  
Oil In Water

Amphipathic enzyme-polymer conjugates were prepared by reaction of hydrophilic enzymes with an anhydride polymer partially substituted with pendant hydrophobic groups. The products formed non-covalent aggregates of high molecular weight, dissociable by non-ionic detergents and urea and subject to additional aggregation at high ionic strength. Oil-in-water emulsions formed in the presence of such conjugates were shown to possess enzyme activity associated with the surface of the water-immiscible liquid. Fluorochrome labelling of conjugates showed that binding occurred at the surface of solvent droplets, and the conjugates were also found to aggregate liposomes.

scholarly journals Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia.

1984 ◽  
Vol 98 (5) ◽  
pp. 1611-1618 ◽  
Author(s):  
K Sutoh ◽  
M Iwane ◽  
F Matsuzaki ◽  
M Kikuchi ◽  
A Ikai
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
High Molecular Weight ◽  
Physarum Polycephalum ◽  
Binding Protein ◽  
High Ionic Strength ◽  
Actin Binding ◽  
Electron Microscopic ◽  
Actin Binding Protein ◽  
Isolation And Characterization

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.

scholarly journals Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum polynucleotide phosphorylase

1971 ◽  
Vol 121 (4) ◽  
pp. 613-620 ◽  
Author(s):  
Pearl I. Peterkin ◽  
P. S. Fitt
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Nucleic Acid ◽  
Halophilic Bacteria ◽  
High Ionic Strength ◽  
Low Molecular Weight ◽  
Polynucleotide Phosphorylase

1. Polynucleotide phosphorylase was purified 200-fold from Halobacterium cutirubrum. 2. It is membrane-associated and can be solubilized by sonication. 3. The purified enzyme requires a high ionic strength for both stability and activity. 4. It is Mn2+-dependent, has all three typical polynucleotide phosphorylase activities and is specific for nucleoside diphosphates. 5. The enzyme is of low molecular weight.

Dissociation of Factor VIII in the Presence of Proteolytic Inhibitors

1978 ◽  
Vol 40 (02) ◽  
pp. 316-325 ◽  
Author(s):  
Ira I Sussman ◽  
Harvey J Weiss
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Direct Evidence ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Bean Trypsin Inhibitor ◽  
Benzamidine Hydrochloride

SummaryWhen gel filtration of factor VIII is performed with buffers of high ionic strength (1.0 M NaCl or 0.25 M CaCl2), the procoagulant activity elutes with proteins of relatively low molecular weight. It has been suggested that in the presence of proteolytic inhibitors, the procoagulant activity would appear at the void volume. To test this hypothesis, chromatography with buffers of high ionic strength was performed in the presence of benzamidine hydrochloride, soy bean trypsin inhibitor, heparin, DFP, and aprotinin. Under all of these conditions, the procoagulant activity continued to elute with proteins of low molecular weight. Similar findings were obtained after chromatographing cryoprecipitate prepared from the plasma of a normal subject who had received heparin. Thus, at present there is no direct evidence to suggest that proteolysis is involved in the dissociation of factor VIII by buffers of high ionic strength.

scholarly journals Production and characterisation of a marine Halomonas surface-active exopolymer

2019 ◽  
Vol 104 (3) ◽  
pp. 1063-1076
Author(s):  
Tony Gutierrez ◽  
Gordon Morris ◽  
Dave Ellis ◽  
Barbara Mulloy ◽  
Michael D. Aitken
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
High Molecular Weight ◽  
Positive Influence ◽  
Contaminated Sites ◽  
Crude Oils ◽  
Magnetic Resonance Spectroscopic Analysis ◽  
Surface Active ◽  
Low Ionic Strength ◽  
Pure Hydrocarbon

AbstractDuring screening for novel emulsifiers and surfactants, a marine gammaproteobacterium, Halomonas sp. MCTG39a, was isolated and selected for its production of an extracellular emulsifying agent, P39a. This polymer was produced by the new isolate during growth in a modified Zobell’s 2216 medium amended with 1% glucose, and was extractable by cold ethanol precipitation. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed P39a to be a high-molecular-weight (~ 261,000 g/mol) glycoprotein composed of carbohydrate (17.2%) and protein (36.4%). The polymer exhibited high emulsifying activities against a range of oil substrates that included straight-chain aliphatics, mono- and alkyl- aromatics and cycloparaffins. In general, higher emulsification values were measured under low (0.1 M PBS) compared to high (synthetic seawater) ionic strength conditions, indicating that low ionic strength is more favourable for emulsification by the P39a polymer. However, as observed with other bacterial emulsifying agents, the polymer emulsified some aromatic hydrocarbon species, as well as refined and crude oils, more effectively under high ionic strength conditions, which we posit could be due to steric adsorption to these substrates as may be conferred by the protein fraction of the polymer. Furthermore, the polymer effected a positive influence on the degradation of phenanthrene by other marine bacteria, such as the specialist PAH-degrader Polycyclovorans algicola. Collectively, based on the ability of this Halomonas high-molecular-weight glycoprotein to emulsify a range of pure hydrocarbon species, as well as refined and crude oils, it shows promise for the bioremediation of contaminated sites.

KINETIC EFFECTS OF HIGH MOLECULAR WEIGHT KININOGEN AND ZINC ON CONTACT ACTIVATION

1987 ◽  
Author(s):  
J D Shore ◽  
D E Day ◽  
S T Olson
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
High Molecular Weight ◽  
Proper Function ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Contact Activation ◽  
Kinetic Effects ◽  
Activation Rates ◽  
Kinetics Of

Previous work in our laboratory showed that Zn2+ enhanced the rate of kallikrein generation by dextran sulfate (DxSO4) in dialyzed normal plasma, but not in Fitzgerald or Hageman prismas. This could be partially explained by a marked effect of Zn2+ on factor XII autoactivation, and our present work involves zinc effects on other reactions of contact activation. At physiological ionic strength (0.15 μ), the kcat/Km for Xlla activation of prekallikrein (PK) was 0.62 μM™1 s™1 which was increased to 4.35 μM™1 s™1 by the presence of 25μg/ml DxSO4. High molecular weight kininogen (HMK) at 40 nM further increased this to 10.8 μM™1 s™1 , and 5 ¼M Zn2+ had no effect. To determine whether these cofactors promote a surface-dependent activation of PK by XIIa under conditions which weaken the protein-surface interactions, the kinetics were examined at 0.3μ. At this ionic strength, kcat/Km was 0.18 μM™1 s™1 and was unchanged by 25μg/ml DxSO4. This was increased to .805 μM™1 s™1 by 150 nM HMK and further increased 10-fold to 8.35 μM™1 s™1 by 10 μM™1 Zn2+ . Qualitative results were obtained at 0.3 μ for the other reciprocal reaction, XII activation by kallikrein (K). To observe XII activation within 2 hours, both 10 μM Zn2+ and 25 μM HMK were essential, indicating that these cofactors have a very large enhancing effect on the kinetics of this reaction. Chromatography of HMWK on DxSO4-agarose ^ljiowed elution of the protein at 0.42 M NaCl in the absence of Zn2+ ,but at 0.88M in its presence, providing evidence that Zn+ markedly increases the affinity of HMK for DxSO4. Our results are consistent with the increased activation rates observed in the presence of Zn2+ and HMK due to enhanced binding affinity of the reacting proteins to surfaces. This is likely to be essential for proper function of the contact system in blood, where many other proteins compete for surface. Supported by USPHS grant HL-25670

scholarly journals Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.

1989 ◽  
Vol 109 (2) ◽  
pp. 549-556 ◽  
Author(s):  
S Citi ◽  
R A Cross ◽  
C R Bagshaw ◽  
J Kendrick-Jones
Keyword(s):  
Enzyme Activity ◽  
Monoclonal Antibodies ◽  
Ionic Strength ◽  
Brush Border ◽  
Active Sites ◽  
High Ionic Strength ◽  
Enzymatic Activities ◽  
Filament Assembly ◽  
Order Of Magnitude ◽  
Low Ionic Strength

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.

scholarly journals The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin.

1976 ◽  
Vol 69 (1) ◽  
pp. 73-89 ◽  
Author(s):  
L G Tilney
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
High Molecular Weight ◽  
Careful Examination ◽  
Filament Formation ◽  
Heavy Meromyosin ◽  
Associated Proteins ◽  
High Molecular Weight Proteins ◽  
Divalent Salts ◽  
Triton X

When echinoderm sperm are treated with the detergent Triton X-100 at pH 6.4 in 10 mM phosphate buffer, the membranes are solubilized, but the actin which is located in the periacrosomal region remains as a phase-dense cup. These cups can be isolated free from the flagella and chromatin and can be solubilized by increasing the pH to 8.0 and by changing the ionic strength and type of buffer used. Since the actin does not exist in the "F" state in unreacted sperm, and since the actin remains as a unit that does not diffuse away, it must be present in the mature sperm in a bound or storage state. The actin is, in fact, associated with a pair of proteins whose mol wt are 250,000 and 230,000. When the isolated cups are digested with trypsin, these high molecular weight proteins are digested, thereby liberating the actin. The actin will polymerize if heavy meromyosin or subfragment 1 is added to a preparation of isolated cups. Evidence is presented that this pair of high molecular weight proteins is similar in molecular weight and properties to erythrocyte spectrin. Attempts at transforming the storage form of actin in the cup into filaments were only moderately successful. The best conditions for filament formation involve incubating the cup in ATP and divalent salts. Careful examination of these cups reveals that the actin polymerized preferentially on either end of oriented filaments that already exist in the cup, indicating that self-nucleation is inefficacious. I conclude that the actin can exist in the storage form by its association with spectrin-like molecules and that the actin in this state polymerizes preferentially onto existing filaments.

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