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L Greene

Publications and source records attributed to L Greene.

At least 37 records · Page 2Linked to original sources
PubMed1995

Polymerization of 70-kDa heat shock protein by yeast DnaJ in ATP.

DnaK, the Escherichia coli hsp70 protein, interacts with DnaJ, a protein cofactor that appears to be involved in presenting protein substrates to DnaK. The yeast DnaJ homolog, YDJ1, has also been shown to interact with yeast hsp70, although the function of this interaction is unknown. In the present study, we investigated the interaction of YDJ1 with both yeast and bovine brain hsp70. We found that, in the presence of ATP, where hsp70 is normally monomeric, YDJ1 induced almost all of the yeast and bovine brain hsp70 to form large polymers, which are readily sedimentable. These polymers were much larger than the dimers and trimers of hsp70, which normally form in the presence of ADP. YDJ1 appeared to be acting catalytically since very little YDJ1 copolymerized with the hsp70, and maximum polymerization occurred at low ratios of YDJ1 to hsp70. The polymerization required ATP and was completely reversed when ATP was replaced by ADP. These data suggest that, in the presence of ATP, YDJ1 may present one hsp70 to another just as under other conditions DnaJ is able to present protein substrates to DnaK.

Adenosine Triphosphate↗
PubMed1995

Interaction of nucleotide-free Hsc70 with clathrin and peptide and effect of ATP analogues.

The functions of the 70 kDa heat-shock proteins (hsp70s) are regulated by their bound nucleotide. We previously observed major differences in the effect of bound ATP and ADP on the interaction of hsc70 (constitutive hsp70) with its protein substrates. In the present study, we investigated the interaction of protein substrates with nucleotide-free hsc70 and with hsc70 with bound ATP analogues. We found, first, that nucleotide-free hsc70 appeared to interact differently with different substrates. Specifically, nucleotide-free hsc70 behaved much more like hsc70-ATP than hsc70-ADP in that clathrin very rapidly bound to and dissociated from nucleotide-free hsc70 in contrast to its very slow binding to and dissociation from hsc70-ADP. On the other hand, nucleotide-free hsc70 behaved more like hsc70-ADP than hsc70-ATP in that cytochrome c peptide dissociated very slowly from nucleotide-free hsc70 compared to its rapid dissociation from hsc70-ATP. Second, binding of the ATP analogues AMP-PNP, dATP, and ATP gamma S to nucleotide-free hsc70 had very little further effect on the properties of the nucleotide-free hsc70. Therefore, previously observed effects of ATP analogues may have been due to removal of the bound ADP rather than to the presence of analogues.

Adenosine Diphosphate↗
PubMed1994

Complex formation between clathrin and uncoating ATPase.

The bovine brain uncoating ATPase, a constitutive 70-kDa heat shock protein, uncoats clathrin-coated vesicles in an ATP-dependent reaction. The uncoating ATPase-clathrin complex formed from the uncoating reaction was compared to the complex formed by directly binding free clathrin to uncoating ATPase. The amount of the latter complex shows a simple hyperbolic dependence on either free clathrin or free uncoating ATPase concentration, whichever is in excess, with a binding stoichiometry of one uncoating ATPase per clathrin heavy chain. ATP markedly increases the rates of formation and dissociation of this complex while ADP profoundly inhibits these rates. At low uncoating ATPase concentrations, much more complex is formed by uncoating than by directly binding clathrin to enzyme. However, during column chromatography or dilution for electron microscopy, both types of complex dissociate in ATP but not ADP, and electron microscopy of both types of complex diluted into ADP shows binding only to the vertex of the clathrin triskelion. We conclude that the uncoating ATPase forms only one type of complex with clathrin and has only one site for nucleotide; ADP at this site prevents either formation or dissociation of complex, whereas ATP at this site allows both processes to occur rapidly.

Adenosine Diphosphate↗
PubMed1994

Characterization of nucleotide-free uncoating ATPase and its binding to ATP, ADP, and ATP analogues.

The interactions of the 70-kDa heat-shock proteins (hsp70s) with their protein substrates appear to be regulated by bound nucleotide. Previous work has shown that the nucleotide binding site of the bovine brain uncoating ATPase, a constitutive member of the hsp70 family, crystallographically resembles the nucleotide binding site of actin and, like actin, the uncoating ATPase has a strongly bound ADP which cannot be removed by dialysis or treatment with ethylenediaminetetraacetic acid (EDTA). This suggests that, like the bound nucleotide of actin, it may be required for the enzyme to retain its native structure. In this study, the strongly bound ADP was removed by first replacing it with 5'-adenylyl imidodiphosphate (AMP-PNP) and then removing the bound AMP-PNP by dialysis. Following this treatment, more than 95% of the uncoating ATPase becomes nucleotide-free. The nucleotide-free uncoating ATPase retains its ability to bind and hydrolyze ATP and to uncoat clathrin-coated vesicles, even after 10 days of storage at 4 degrees C. Therefore, in contrast to actin, the bound nucleotide of the uncoating ATPase is not required to prevent denaturation of the enzyme. Using nucleotide-free uncoating ATPase, we were able to accurately measure the dissociation constants of ATP, ADP, and the nucleotide analogues AMP-PNP and 2'-deoxyadenosine 5'-triphosphate (dATP). The dissociation constants of both ATP and ADP are about 10(-8) M, more than 1-2 orders of magnitude stronger than previously reported, while AMP-PNP and dATP bind 2-3 orders of magnitude more weakly than ATP.

Adenosine Diphosphate↗
PubMed1993

A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase.

Immediately after clathrin-coated pits pinch off from the cell membrane to form clathrin-coated vesicles, clathrin dissociates from the vesicles. In vitro studies suggest that this dissociation is carried out by the uncoating ATPase, a constitutive member of the 70-kDa heat shock family. Aside from the requirement for ATP, nothing is known about the regulation of the uncoating process. We now show that clathrin baskets prepared from highly purified clathrin and AP2, the assembly protein associated with plasma membrane coated vesicles, cannot be uncoated by the bovine brain uncoating ATPase alone. A 100-kDa protein cofactor, which was isolated from coated vesicles, is essential for uncoating by the uncoating ATPase. This cofactor restores normal uncoating when present at a molar ratio of about 1 to 10 to clathrin and uncoating ATPase.

Adenosine Triphosphatases↗
PubMed1993

Nucleotide binding properties of bovine brain uncoating ATPase.

Many functions of the 70-kDa heat-shock proteins (hsp70s) appear to be regulated by bound nucleotide. In this study we examined the nucleotide binding properties of purified bovine brain uncoating ATPase, one of the constitutively expressed members of the hsp70 family. We found that uncoating ATPase purified by ATP-agarose column chromatography retained one ADP molecule bound per enzyme molecule which could not be removed by extensive dialysis. Since this bound ADP exchanged rapidly with free ADP or ATP, the inability to remove the bound nucleotide was not due to slow dissociation but rather to strong binding of the nucleotide to the uncoating ATPase. In confirmation of this view, equilibrium dialysis experiments suggested that the dissociation constants for both ADP and ATP were less than 0.1 microM. Schmid et al. (Schmid, S. L., Braell, W. A., and Rothman, J. E. (1985) J. Biol. Chem 260, 10057-10062) suggested that the uncoating ATPase had two sites for bound nucleotide, one specific for ATP and one binding both ATP and ATP analogues but not ADP. In contrast, we found that enzyme with bound ADP did not bind further adenosine 5'-(beta,gamma-imino)triphosphate or dATP, nor did more than one ATP molecule bind per enzyme even in 200 microM free ATP. These results strongly suggest that the enzyme has only one binding site for nucleotide. During steady-state ATP hydrolysis, 85% of the bound nucleotide at this site was determined to be ATP and 15% ADP; this is consistent with the rate of ADP release determined in the exchange experiments noted above, where ADP release was found to be six times faster than the overall rate of ATP hydrolysis.

Adenosine Diphosphate↗
PubMed1986

Changes in antipyrine and indocyanine green kinetics during nifedipine, verapamil, and diltiazem therapy.

Ten healthy subjects received oral antipyrine and intravenous indocyanine green (ICG) alone and after 5 days of oral nifedipine, diltiazem, and verapamil. Antipyrine clearance decreased during verapamil (range 4% to 26%) and diltiazem (6% to 24%) therapy (P less than 0.001) but did not change during nifedipine treatment. Antipyrine t1/2 also increased during verapamil and diltiazem treatment (P less than 0.001). ICG clearance did not change during diltiazem therapy but increased during dosing with nifedipine and verapamil (P less than 0.05). Estimated liver blood flow (derived from ICG clearance and hematocrit) also increased during verapamil (mean 33%) and nifedipine (mean 27%) treatment (P less than 0.05). Drug interactions with other liver-metabolized drugs may occur during therapy with these calcium antagonists. Nifedipine appears to increase liver blood flow whereas diltiazem inhibits oxidative drug metabolism. Drug interactions with verapamil could involve both mechanisms.

Adult↗
PubMed1985

Epinephrine-induced cyclic AMP production in skin fibroblasts from patients with dementia of Alzheimer type and controls.

Skin biopsies were obtained from six patients with dementia of Alzheimer type (DAT) and from three hospitalized age matched controls. Fibroblasts from these biopsies were grown in culture and compared for their growth characteristics and sensitivity to epinephrine with four cultures from age matched healthy individuals. The growth characteristics were similar in all three groups. The basal levels of cyclic AMP and the epinephrine-induced increase of cyclic AMP levels were also similar in control and DAT cells.

Aged↗