Research In Biochemistry


Research In Biochemistry Essay, Research Paper

Angela LucasBiochemistry 3334/16/99Tullberg, A., Hakansson, G., and H. Race. 1998. A protein tyrosine kinase of chloroplast thylakoid membranes phosphorylates light harvesting complex II proteins. Biochemical and Biophysical Research Communications 250: 617-622. As we have studied in just about every system and pathway learned in Biochemistry 333, reversible phosphorylation of biological molecules is a key regulatory mechanism for cell function. Kinases are proteins that transfer a phosphate group from ATP to another protein to activate it, inactivate it, cause a conformational change, etc. In particular, protein tyrosine kinases (PTKs) transfer a phosphate group from ATP to a tyrosine residue of a protein. Although most studies of PTKs have been done with animal cells, new evidence suggests that PTKs may also be present in photosynthetic bacteria and possibly plants. Phosphotyrosine residues isolated from phytochrome indicates that these plants may use light-regulated PTKs. To determine whether there is PTK activity in thylakoid membranes, the researchers employed three different approaches to identify both the enzymatic activity and its substrates. First, in vitro protein phosphorylation was analyzed in the presence of genistein, a specific tyrosine kinase inhibitor. To do this, chloroplasts taken from pea plant leaves, containing thylakoid membranes, were combined with ATP to allow phosphorylation to occur. Genistein was then added and the rates of phosphorylation measured before and after. It was found that increasing concentrations of genistein correlated directly with a decrease in phosphorylation in nine thylakoid phosphoproteins. In addition, three phosphoproteins were identified using immunological techniques : an 11 kDa protein, a 27kDa protein, and a 29kDa protein, with the larger two being components of light-harvesting complex II. Next, the stability of in vitro phosphorylated amino acids was examined to identify possible substrates of the PTK. To determine this, SDS-PAGE was performed with the proteins, which were then transferred to a PVDF membrane. The proteins were then exposed to acid/alkaline washes. At a high pH, the phosphate ester bond to both threonine and serine residues is unstable, whereas phosphotyrosine remains stable. So exposure to the alkaline wash would break down a majority of the phosphothreonine and phosphoserine bonds while leaving phoshotyrosine intact. Analysis after the acid/alkaline wash showed that the light-harvesting proteins, in particular, retained a majority of their phosphate bond, indicating the presence of phosphotyrosine. The conclusion from this is that the light-harvesting complex II proteins are substrates for the PTK. Lastly, a monoclonal antibody specific for phosphotyrosine residues was employed to confirm the presence of these residues in specific proteins. The antibodies were applied to the proteins which had been transferred to nitrocellulose paper and immunoreactions visualized. Through this technique, nine proteins were found to react with the anti-phosphotyrosine antibodies. Very strong immunoreactions were seen with two proteins in particular: a 27 and a 29 kDA protein, most likely components of the light-harvesting complex II. These results support the conclusion that the PTK activity is directed toward the phosphorylation of the light-harvesting complex II proteins of the chloroplast thylakoid membrane.

Додати в блог або на сайт

Цей текст може містити помилки.

A Free essays | Essay
4.6кб. | download | скачати

Related works:
Biochemistry Lab
Stages and types of an applied sociological research Sociological research process
A And P Research
How To Writing A Research
Animal Research
© Усі права захищені
написати до нас