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Japanese radish tuber and leaf produced antifreeze proteins (AFPs) having thermal hysteresis activity (TH) and ice recrystallization inhibiting activity (RI). Upon cold acclimation, the apoplastic fluid of the Japanese radish exhibited hexagonal crystal growth, indicating the presence of an antifreeze protein. The induction patterns of protein and the TH activity of apoplastic fraction from both samples were different. The TH activities of apoplastic fraction from tuber and leaves were 0.20 +/- 0.03 and 0.18 +/- 0.02 degree C, respectively. Also, the TH and RI activities of apoplastic fraction of leaves were activated by autoclave treatment at pH 10.0. An antifreeze peptide (molecular weight 1320), was purified using chromatography. Furthermore, the chitinase and beta-1, 3-glucanase activities in the apoplastic fraction of its tuber were induced by the cold acclimation. Some proteins in this apoplastic fraction were reacted with the anti-glucanase-like protein (GLP) antiserum and anti-chitinase-like protein (CLP) antiserum produced against isolated winter rye AFPs. This is the first report on the presence and characterization of AFPs from Japanese radish tuber.

One strain of Antarctic bacteria, Flavobacterium xanthum IAM12026, has a highly active antifreeze protein (AFP) in the intracellular space. The cell-free extract from strain IAM12026 after culturing at 4 degree C for 7 days in TSB medium, had activity of 0.04 degree C at a concentration of 0.7 mg/ml. The ice crystals formed do not have distinct facets without typically rounded shape and the changes of their morphology during the course of the thermal hysteresis (TH) measurement. The ice crystal 'burst' occurring at the end-point of the TH is dendritic with hexagonal symmetry. Also, this activity was not affected by the treatment of dialysis and the addition of EDTA. Furthermore, this cell-free extract had high levels of ice recrystallization-inhibiting (RI) activity like those of Fish AFPs. The AFP (FlAFP) was homogeneity purified using chromatography. A relative molecular mass of approximately 59,000 was calculated from gel filtration and SDS-PAGE data. The thermal stability of FlAFP was below 50 degree C, and TH value was absent above 60 degree C. The TH value of FlAFP was activated at 5.2 degree C by the addition of 0.5 M malate. This activation was decreased with increasing protein concentration. To our knowledge this is the first report on the high level of TH and RI activities of bacterial intracellular AFP.

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.

We found six bacteria capable of producing antifreeze protein (AFP) from Ross Island, Antarctica. Among these AFP-producing bacteria, strain No. 82 had the highest antifreeze activity and was identified as Moraxella sp. The optimum temperature and pH for the production of AFP were 5 degrees C and 7.0, respectively. After partially purifying the AFP from the culture supernatant using 60% saturation of ammonium sulfate, only the 52-kDa protein band (100 microg/ml) which eluted from SDS-PAGE indicated antifreeze activity by the formation of hexagonal crystals. Furthermore, we confirmed that this AFP was a lipoprotein by the lipid stain test and treatment with some enzymes and that it had no ice-nucleating activity. Also, the N-terminal amino acid sequence of this AFP had high similarity with that of outer membrane proteins from Moraxella (Branhamella) catarrhalis. This is the first report of AFP-producing bacteria in Antarctica and an antifreeze lipoprotein (AFLP) from Moraxella sp.

Investigates type II antifreeze protein (AFP) from a mid-latitude freshwater fish, the Japanese smelt or Hypomesus nipponensis. Purification and characterization of Japanese smelt AFP; N-terminal DNA sequence of Japanese smelt AFP.