A regiospecific synthesis of branched tetranucleotides: U3'p5'A2'p5'G 3'p5'U and U3'p5'A2'p5'G3'p5'C

An efficient strategy for the synthesis of branched nucleotides 14 and 15 has been developed using key intermediates 6 and 10.     

Functional specificity of guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases from silkworm.

Adenosine 3':5'-monophosphate-dependent protein kinase partially purified from silkworm pupae shows identical functional activities with those of mammalian protein kinases; the insect and mammalian kinases are completely exchangeable in the phosphorylation of muscle glycogen phosphorylase kinase and glycogen synthetase resulting in the activation and inactivation of the respective enzymes. In contrast, guanosine 3':5'-monophosphate-dependent protein kinase obtained from the same organism is totally inactive in this role and phosphorylates different, mainly seryl and some threonyl, residues of acceptor proteins. Substrates of the latter kinase intimately involved in the regulation of biological processes have remained unknown.     

Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.

In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.     

Characterization of calf-ovary adenosine;3':5'-monophosphate-dependent protein kinases and adenosine-3':5'-monophosphate-binding proteins.

Protein phosphokinase activity from the calf ovary cytosol (105000 X g supernatant fraction) has been resolved by chromatography and polyacrylamide gel electrophoresis into two major protein kinases, PK-H1 and PK-H2, both dependent on adenosine 3':5'-monophosphate (cyclic AMP). The enzymes have similar molecular weights (230000) and substrate specificities but differ in their cyclic-AMP-dependency and stimulation by cyclic AMP. The differences have been explained by the presence in PK-H1 of a unique cyclic-AMP-binding protein which has little catalytic activity associated with it. The cyclic-AMP-binding protein has a high affinity for cyclic AMP and in addition is able to inhibit the activity of the isolated catalytic subunit. The ovarian cyclic-AMP-dependent protein kinases have properties similar to those found in other tissues. They can be dissociated into catalytic and regulatory subunits and are inhibited by a heat-stable protein inhibitor isolated from rabbit skeletal muscle. Preincubation of the cytosol with high levels of cyclic AMP resulted in additional cyclic-AMP-dependent protein kinases and cyclic-AMP-binding proteins which include protein kinases and binding proteins of greater than 400 000 molecular weight.     

The penetration of 3:5-dinitro-o-cresol into leaves

A study of the penetration of 3:5-dinitro- 0 -cresol from spray solutions or suspensions into leaves of Sinapis arvensis L. shows that entry through the stomata is unimportant. Lethal amounts may enter from aqueous solution by simple diffusion of the undissociated phenol through the epidermis, which behaves as a homogeneous lipide membrane. The effect of the structure of the epidermal surface on the rate of diffusion is discussed. Lethal amounts may also enter Sinapis from a dry surface deposit by gaseous diffusion through the stomata or by diffusive penetration of the epidermis following sublimation. Movement of the substance within a plant is slight and appears to be mainly by diffusion. The selective action of 3:5-dinitro- 0 -cresol as a weed-killer is discussed in the light of these results.