The four-step caffeine biosynthetic pathway includes three methylation steps that utilise S-adenosyl-L-methionine (SAM) as the methyl donor. In the process SAM is converted to S-adenosyl-L-homocysteine (SAH) which in turn is hydrolysed to L-homocysteine and adenosine. Significant amounts of radioactivity from [methyl-14 C]methionine and [methyl-14 C]SAM were incorporated into theobromine and caffeine in young tea leaf segments, and very high SAH hydrolase activity was found in cell-free extracts from young tea leaves. Substantial amounts of radioactivity from [adenosyl-14 C]SAH were also recovered as theobromine and caffeine in tea leaf segments, indicating that adenosine derived from SAH is utilised for the synthesis of the purine ring of caffeine. From the profiles of activity of related enzymes in tea leaf extracts, it is proposed that the major route from SAM to caffeine is a SAMC CSAHC CadenosineC CadenineC CAMPC CIMPC CXMPC CxanthosineC C7-methylxanthosineC C7-methylxanthineC CtheobromineC Ccaffeine pathway. In addition, direct adenosine kinase-catalysed formation of AMP from adenosine may participate as an alternative minor route. The activity of two of the three N-methyltransferase activities involved in caffeine biosynthesis and part of the activities of SAH hydrolase, adenosine nucleosidase, adenine phosphoribosyltransferase and adenosine kinase were located in tea chloroplasts. In contrast, no detectable activity of SAM synthetase was associated with the purified chloroplast fraction. This is a first demonstration that the purine skeleton of caffeine is synthesised from adenosine released from the SAM cycle. ß

Profiles of nucleotide levels in two varieties of Japanese green teas (cv. Yabukita and Saemidori), a Chinese green tea (Longjing), and two Japanese black teas (cv. Benifuuki and Benihikari) were determined and compared with that of fresh tea leaves. The concentration of 5'-nucleotides in green tea was much higher than in black tea. Nucleoside diphosphates were present in larger amounts than nucleoside triphosphates in manufactured green and black teas, whereas the triphosphates predominated in fresh tea leaves. Low levels of 3'-nucleotides were found in green and black teas. Inosine 5'-monophosphate, which is utilized as a seasoning component, was found in all manufactured teas in concentrations ranging from 50 to 200 nmol/g of dry weight. The levels of both inosine 5'-monophosphate and guanosine 5'-monophosphate were high in Chinese Longjing green tea. The unique profiles of nucleotides in manufactured teas may be a consequence of the action of degradation enzymes, such as ribonuclease, apyrase, phosphatase, nucleotidase, and adenosine 5'-monophsphate deaminase during the commercial processing of the young leaves.

Potato Tuber, PFP, Fructose-2,6-bisphosphate A seven fold increase in the rate of respiratory 0 2 uptake was observed 24 h after slicing of potato tuber disks. The maximum activity of pyrophosphate:fructose-6-phosphate phos photransferase (PFP) was 5 -7 times greater than that of A TP-dependent phosphofructokinase (PFK) in fresh or aged potato slices. Thus, PFP may participate in glycolysis which supplies respiratory substrate in potato tubers. The PFP activity of desalted extracts deter mined in the absence of fructose-2,6-bisphosphate (F2,6BP) increased by 4.5 fold 24h after slicing. However, maximal PFP activity determined with saturating (1 ^.m) F2.6BP was not changed. The K a values of PFP for F2,6BP was lowered from 33 to 7 nM after 24 h of aging treatm ent. This increased susceptibility of the PFP activity to its allosteric activator, F2,6BP, may be involved in the increased respiration in wounded disks of potato tubers. Imunoblotting experiments indicated that both the a (66 kDa) and the ß (60kDa) subunits of PFP were present in fresh or 24h aged tuber slices.