Approaches to Disulfide Bonds Identification in the Na+/K+-ATPase Alpha Subunit

1994 ◽  
pp. 374-376
Author(s):  
N. M. Gevondyan ◽  
N. N. Modyanov
Keyword(s):  
Disulfide Bonds ◽  
Alpha Subunit

scholarly journals Functional significance of the beta-subunit for heterodimeric P-type ATPases.

1995 ◽  
Vol 198 (1) ◽  
pp. 1-17 ◽  
Author(s):  
D C Chow ◽  
J G Forte
Keyword(s):  
Cation Exchange ◽  
Intracellular Trafficking ◽  
Disulfide Bonds ◽  
Basolateral Membrane ◽  
Extracellular Domain ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Alpha Beta ◽  
The Stability ◽  
P Type

We have reviewed the structural and functional role of the beta-subunit in a subfamily of the P-ATPases known as the alpha/beta-heterodimeric, cation-exchange ATPases. The subfamily consists of the various isoforms of Na+/K(+)-ATPase and H+/K(+)-ATPase, both of which pump a cation out of the cell (Na+ or H+, respectively) in recycle exchange for K+. Much of the earlier work has emphasized the functional activities of the alpha-subunit, which shares many characteristics with the broader P-ATPase family. It is now clear that the glycosylated beta-subunit is an essential component of the cation-exchange ATPase subfamily. All beta-subunit isoforms have three highly conserved disulfide bonds within the extracellular domain that serve to stabilize the alpha-subunit, alpha/beta interaction and functional activity of the holoenzyme. Evidence strongly suggests that the beta-subunit is involved in the K(+)-dependent reactions of the enzymes, such as the E1-E2 transition and K+ occlusion, and that the extracellular domain of the beta-subunit plays an important role in determining the kinetics of K+ interaction. In most vertebrate cells, the unassociated alpha-subunit is restricted to the endoplasmic reticulum (ER), and assembly of the alpha/beta complex occurs within the ER. Signals for exiting the ER and directing the correct intracellular trafficking are primarily determined by the beta-subunit; Na+/K(+)-ATPase typically terminates in the plasma membrane facing the basolateral membrane, whereas all isoforms of H+/K(+)-ATPase terminate in the apical membrane. The C-terminal extracellular domain of the beta-subunit is important for proper interaction with the alpha-subunit and for correct intracellular trafficking. Oligosaccharides on the beta-subunit are not essential for enzyme function, but do serve to enhance the efficiency of alpha/beta association by increasing the lifetime of the unassociated beta-subunit and the stability of the alpha/beta complex to tryptic attack. We propose that highly specialized glycosylation on the beta-subunit of the gastric H+/K(+)-ATPase may help to protect that enzyme from the harsh extracellular environment of the stomach.

Gastric H(+)-K(+)-ATPase activity is inhibited by reduction of disulfide bonds in beta-subunit

1992 ◽  
Vol 263 (1) ◽  
pp. C39-C46 ◽  
Author(s):  
D. C. Chow ◽  
C. M. Browning ◽  
J. G. Forte
Keyword(s):  
Atpase Activity ◽  
Disulfide Bonds ◽  
Elevated Temperatures ◽  
Sequence Data ◽  
Kinetic Studies ◽  
Monovalent Cation ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Cysteine Residues ◽  
High Concentrations

H(+)-K(+)-ATPase activity of rabbit isolated gastric microsomes was irreversibly inactivated by reducing agents, such as 2-mercaptoethanol and dithiothreitol. Similar to what has been observed for Na(+)-K(+)-ATPase, high concentrations of reagents, at moderately elevated temperatures, were required to inactivate H(+)-K(+)-ATPase, suggesting relative inaccessibility of the responsible disulfide bonds. Resistance against inactivation was conferred by monovalent cation activators of K(+)-stimulated ATPase and p-nitro-phenylphosphatase. The effectiveness of K+ congeners in protecting the enzyme was similar in sequence (Tl+ greater than K+ greater than Rb+) and concentration to their respective affinities for stimulating enzymatic activity, suggesting that the K(+)-bound form of the enzyme is more resistant to reduction than the free enzyme. Furthermore, Na+ antagonized the protective effect of K+. Labeling studies using fluorescein-maleimide indicated that 60-70% of the cysteine residues in the beta-subunit are in the oxidized form. Coupled with primary sequence data, this suggests that three disulfide bonds are present in the native beta-subunit. In contrast, less than 10% of the cysteine residues in the alpha-subunit are in the oxidized form. Kinetic studies showed that the 2-mercaptoethanol-induced loss of H(+)-K(+)-ATPase activity was correlated with a reduction of disulfide groups in the beta-subunit, while there was no significant change in the alpha-subunit. We conclude that reduction of disulfide bonds irreversibly inhibits H(+)-K(+)-ATPase activity, binding of K+ to the enzyme confers a resistance to disulfide bond reduction, and the responsible disulfide bonds are present in the beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Mutagenesis of cysteine residues in the human gonadotropin alpha subunit. Roles of individual disulfide bonds in secretion, assembly, and biologic activity.

1994 ◽  
Vol 269 (41) ◽  
pp. 25543-25548 ◽  
Author(s):  
M Furuhashi ◽  
H Ando ◽  
M Bielinska ◽  
M R Pixley ◽  
T Shikone ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Alpha Subunit ◽  
Cysteine Residues ◽  
Biologic Activity

Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

1992 ◽  
Vol 50 (1) ◽  
pp. 530-531
Author(s):  
P. F. Flicker ◽  
V.S. Kulkarni ◽  
J. P. Robinson ◽  
G. Stubbs ◽  
B. R. DasGupta
Keyword(s):  
Disulfide Bonds ◽  
Clostridium Botulinum ◽  
Structural Changes ◽  
Two Dimensional ◽  
Type B ◽  
Single Chain ◽  
Muscle Paralysis ◽  
Cryo Electron Microscopy ◽  
Disulfide Linkages ◽  
Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

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