ADENYLATE KINASE IN TOBACCO CELL-CULTURES .1. SEPARATION AND LOCALIZATION OF DIFFERENT ACTIVITIES

Adenylate kinase (AK, EC 2.7.4.3) activity and its compartmentation we re investigated in cell cultures of Nicotiana tabacum L. cv. 'Havana 4 25'. An electrophoretic pattern consisting of six bands was detected b y isoelectrofocusing-polyacrylamide gel electrophoresis (IEF-PAGE) and native stain procedures for both directions of the AK-catalysed react ion. Controls using the specific inhibitor adenosine(5')pentaphospho(5 ')adenosine or omitting AMP in the ATP/AMP-stain were performed in ord er to ascertain that the staining was not due to phosphatases or other interfering activities. Mitochondria (80-95% intact) were isolated fr om cell suspension culture of the line CEH-3. IEF-PAGE revealed only o ne activity band with a pI of 7.5. Latency tests have shown that this activity is located in the intermembrane space. Chloroplasts (75-85% i ntact) were isolated from fully grown tobacco leaves. Three activity b ands were found in IEF-PAGE with pI of about 4.55, 4.50 and 4.45 respe ctively. Latency tests indicated different locations within the chloro plast. If plastid morphogenesis in calli was suppressed by norflurazon e, all three chloroplast bands lacked. The mitochondrial band and, esp ecially, the band at pI 4.60 were enhanced while the band at pI 4.35 w as unchanged. This result confirms the established localisation and su ggests the pI 4.60-band as a cytosolic activity.