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1.
State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using saturation-transfer electron spin resonance 总被引:1,自引:0,他引:1
The state of aggregation of the (Ca2+ + Mg2+)-ATPase in the membrane of sarcoplasmic reticulum and in reconstituted membrane systems has been studied using saturation-transfer electron spin resonance (ST-ESR). Saturation-transfer ESR spectra show that in the sarcoplasmic reticulum, the ATPase is relatively free to rotate, with an effective rotational correlation time of approx. 33 microseconds at 4 degrees C, consistent with a monomeric or dimeric structure. The rate of rotation is observed to decrease with decreasing molar ratio of lipid to protein. In reconstituted systems, rotational motion of the ATPase on the millisecond time scale ceases when the lipids are in the gel phase. Addition of decavanadate, which causes the formation of crystalline arrays in negatively stained electron micrographs, results in only a small reduction in rotation rate for the ATPase in the membrane. The experiments are interpreted in terms of a short-lived (on the millisecond time scale) protein-protein interaction, with the formation of crystalline clusters of ATPase molecules which form and melt rapidly. 相似文献
2.
Corticosterone, the major stress hormone, plays an important role in regulating neuronal functions of the limbic system, although the cellular targets and molecular mechanisms of corticosteroid signaling are largely unknown. Here we show that a short treatment of corticosterone significantly increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission and AMPAR membrane trafficking in pyramidal neurons of prefrontal cortex, a key region involved in cognition and emotion. This enhancing effect of corticosterone is through a mechanism dependent on Rab4, the small GTPase-controlling receptor recycling between early endosome and plasma membrane. Guanosine nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab proteins between membrane and cytosol, forms an increased complex with Rab4 after corticosterone treatment. Corticosterone also triggers an increased GDI phosphorylation at Ser-213 by the serum- and glucocorticoid-inducible kinase (SGK). Moreover, AMPAR synaptic currents and surface expression and their regulation by corticosterone are altered by mutating Ser-213 on GDI. These results suggest that corticosterone, via SGK phosphorylation of GDI at Ser-213, increases the formation of GDI-Rab4 complex, facilitating the functional cycle of Rab4 and Rab4-mediated recycling of AMPARs to the synaptic membrane. It provides a potential mechanism underlying the role of corticosteroid stress hormone in up-regulating excitatory synaptic efficacy in cortical neurons. 相似文献
3.
Receptors for plant growth regulators: Recent advances 总被引:7,自引:0,他引:7
We have reviewed recent progress in research on plant growth regulator (PGR) receptors. For some growth regulators, no receptor protein has yet been identified, but promising new approaches are discussed. For other receptors, specific and sensitive probes have been developed and, in one case, the membrane-associated auxin-binding protein of maize, these have been used to study the function of the receptor. The maize receptor has been cloned and sequenced; cDNA probes will allow the expression of receptor genes in normal and transformed plants to be studied. PGR sensitivity mutants have been described and, in conjunction with biochemical probes, should prove valuable in elucidating the functions of receptors and the nature of subsequent signal transduction events. 相似文献
4.
Richard M. Napier Michael A. Venis Michael A. Bolton Louise I. Richardson Goeffrey W. Butcher 《Planta》1988,176(4):519-526
Binding proteins, thought to be auxin receptors, can be solubilised from maize (Zea mays L.) membranes after acetone treatment. From these crude extracts, receptor preparations of over 50% purity can be obtained by a reliable, straight-forward procedure involving three chromatographic steps — anion exchange, gel filtration and high-resolution anion exchange. Such preparations have been used to immunise rats for subsequent production of monoclonal antibodies. By the further step of native polyacrylamide gel electrophoresis the semi-purified preparations yield homogeneous, dimeric (22-kilodalton, kDa) auxin-binding protein, which has been used to produce a polyclonal rabbit antiserum. The preliminary characterisation of this antiserum and of the five monoclonal antibodies is presented. Two of the monoclonal antibodies specifically recognise the major 22-kDa-binding protein polypeptide whilst the other three recognise, in addition, a minor 21-kDa species. All the monoclonal antibodies recognise the polypeptide rather than the glycan side chain and the polyclonal antiserum also recognises deglycosylated binding protein. The antibodies have been used to quantify the abundance of auxinbinding protein in a number of tissues of etiolated maize seedlings. Root membranes contain 20-fold less binding protein than coleoptile membranes.Abbreviations ABP
auxin-binding protein
- DEAE
diethylaminoethyl
- Ig
immunoglobulin
- kDa
kilodalton
- NAA
naphthalene-1-acetic acid
- Mr
relative molecular mass
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl sulfate 相似文献
5.
D L Milton M L Napier R M Myers J K Hardman 《The Journal of biological chemistry》1986,261(35):16604-16615
A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the Escherichia coli tryptophan synthase. A random single base pair saturation mutagenesis procedure (Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 229, 242-247) was applied in vitro to subcloned fragments of the trpA gene, which codes for this polypeptide. Mutagenesis plasmid vectors were constructed containing three fragments of the trpA gene which together code for about half of the total amino acid residues of the alpha-subunit. The vectors were constructed such that each strand of each trpA fragment could be altered. These trpA fragments were mutagenized in vitro (using nitrous acid, formic acid, hydrazine, and potassium permanganate), and several thousands of mutants have been isolated. Thirty-two mutants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the alpha-subunit) have been sequenced. Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alterations resulted in single amino acid substitutions. Selected mutant trpA fragments were subcloned into an overexpression vector in which the trpA gene is controlled by the tac promoter and is inducible by lactose. The kinetics and extent of induction show that after 22 h of induction, the wild-type alpha-subunit constituted about 30% of the total protein. A simple one-step purification procedure for the alpha-subunit is described in which 15 mg of alpha-subunit can be obtained from 200 ml of fully induced cultures. The mutant trpA genes were induced for mutant alpha-subunit expression, and an initial examination of their properties in crude extracts was performed. Of the 17 mutant proteins examined, most were overproduced to levels comparable to that for the wild-type alpha-subunit. An analysis of the apparent stability, beta 2-subunit-activating activity, and intrinsic activity of this group of mutant alpha-subunits suggests that many amino acid alterations have no apparent effect; there is a variety of novel functional defects; and a sequence located near residues 28 through 54 may be particularly critical for the normal folding of the polypeptide. 相似文献
6.
Microinjection of neurotensin (NT; 2 and 5 μg) into the substantia nigra zona compacta caused an increase in dopamine (DA) and DA metabolites in the rodent globus pallidus and striatum which persisted for at least 20 hours after peptide administration. Similar NT treatments given unilaterally into the nigra caused circling away from the injected side in amphetamine-pretreated rats, but were without effect when microinjected into saline-pretreated animals. Circling also occurred when the animals were given amphetamine 20 hours after intranigral NT administration. Contralateral rotation was observed with unilateral intranigral injections of gamma-hydroxybutyric acid (GHB; 400 μg) or with lower intranigral GHB doses (250 μg) in amphetamine-pretreated animals. The effects of GHB and NT differed in the manner in which the animals rotated as well as in the profile of DA and DA metabolite changes induced by these drugs. These studies indicated that: (1) dopaminergic functions of the globus pallidus are influenced, like the striatum, by manipulations of the substantia nigra; (2) NT and GHB likely act via different mechanisms to effect nigral dopamine-containing cells; and (3) NT was capable of inducing changes in dopamine neurons which had long term consequences. 相似文献
7.
Otto D. Hensens Ray S. Dewey Jerrold M. Liesch Mary A. Napier Robert A. Reamer Jack L. Smith Georg Albers-Schönberg Irving H. Goldberg 《Biochemical and biophysical research communications》1983,113(2):538-547
Spectroscopic evidence suggests the presence of a highly strained ether ring (Fig. 1) (possibly an epoxide) in the C12-subunit of the previously determined partial structure (Fig. 2) of the major neocarzinostatin chromophore (NCS-Chrom ) which completes assignment of all the oxygens in the molecule. The main product from mercaptan treatment suggests opening of the ether ring involving the addition of one molecule of mercaptan as well as reduction of the C12-substructure, whereas a parallel two-step reduction occurs on NaBH4 treatment. Both reactions occur with rearrangement of the C12-substructure and the implication for the mechanism of action of NCS-Chrom in DNA strand scission activity is discussed. The evidence suggests a downward revision of the molecular formula for NCS-Chrom as well as minor components and by two protons. 相似文献
8.
9.
Barindra Sana Ke Ding Jia Wei Siau Rupali Reddy Pasula Sharon Chee Sharad Kharel Jean-Baptise Henri Lena Eunice Goh Lakshminarayanan Rajamani Yeng Ming Lam Sierin Lim John F. Ghadessy 《Biotechnology and bioengineering》2023,120(11):3200-3209
Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity. 相似文献
10.