scholarly journals Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota)

1999 ◽  
Vol 340 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Maggie SMALLWOOD ◽  
Dawn WORRALL ◽  
Louise BYASS ◽  
Luisa ELIAS ◽  
David ASHFORD ◽  
...  
Keyword(s):  
Plant Extracts ◽  
Daucus Carota ◽  
Thermal Hysteresis ◽  
Antifreeze Protein ◽  
Ice Recrystallization ◽  
Thermal Hysteresis Activity ◽  
Isolation And Characterization ◽  
Tap Root ◽  
Unequivocal Identification

A modified assay for inhibition of ice recrystallization which allows unequivocal identification of activity in plant extracts is described. Using this assay a novel, cold-induced, 36 kDa antifreeze protein has been isolated from the tap root of cold-acclimated carrot (Daucus carota) plants. This protein inhibits the recrystallization of ice and exhibits thermal-hysteresis activity. The polypeptide behaves as monomer in solution and is N-glycosylated. The corresponding gene is unique in the carrot genome and induced by cold. The antifreeze protein appears to be localized within the apoplast.

scholarly journals Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota)

1999 ◽  
Vol 340 (2) ◽  
pp. 385 ◽  
Author(s):  
Maggie SMALLWOOD ◽  
Dawn WORRALL ◽  
Louise BYASS ◽  
Luisa ELIAS ◽  
David ASHFORD ◽  
...  
Keyword(s):  
Daucus Carota ◽  
Antifreeze Protein ◽  
Isolation And Characterization

Effects of antifreeze proteins on red blood cell survival during cryopreservation.

1996 ◽  
Vol 199 (9) ◽  
pp. 2071-2076
Author(s):  
H Chao ◽  
P L Davies ◽  
J F Carpenter
Keyword(s):  
Blood Cell ◽  
Cell Survival ◽  
Red Blood Cell ◽  
Blood Cells ◽  
Thermal Hysteresis ◽  
Relative Efficacy ◽  
Ice Recrystallization ◽  
Type Iii ◽  
Thermal Hysteresis Activity ◽  
Mutant Forms

Antifreeze protein (AFP) types, I, II and III were tested for their ability to protect red blood cells from lysis during warming, after cryopreservation in hydroxyethyl starch. All three types reduced hemolysis to 25% of control values at similar micromolar concentrations but enhanced lysis as the AFP concentration approached millimolar levels. Site-directed mutants of type III AFP with different thermal hysteresis activities were tested for their ability to protect the cryopreserved cells from lysis. Their relative efficacy in protecting the cells correlated closely with their thermal hysteresis activity. Cryomicroscopy indicated that the protection of red cells by type III AFP and the mutant forms was due to inhibition of ice recrystallization.

scholarly journals Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein

2004 ◽  
Vol 377 (3) ◽  
pp. 589-595 ◽  
Author(s):  
Dang-Quan ZHANG ◽  
Bing LIU ◽  
Dong-Ru FENG ◽  
Yan-Ming HE ◽  
Shu-Qi WANG ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Tandem Repeat ◽  
Thermal Hysteresis ◽  
Three Dimensional ◽  
Antifreeze Protein ◽  
Three Dimensional Model ◽  
Prism Plane ◽  
Thermal Hysteresis Activity ◽  
Ice Binding

The ~24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop β-helix containing the ~24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each β-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face.

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