The cytochemical localization of adenylate cyclase: fact or artifact?

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et al. (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin. NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [alpha32P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complet Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is to low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.     

Studies on (K+ + H+)-ATPase V. Chemical composition and molecular weight of the catalytic subunit

(1) A (K+ + H+)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The Detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, N = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S. E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Rat pancreas adenylate cyclase. VI. Role of the enzyme in secretin stimulated enzyme secretion.

1. The responsiveness of adenylate cyclase and enzyme secretin for secretin and the C-terminal octapeptide of pancreozymin has been investigated in particulate fractions of the pancreas of five different species. 2. The adenylate cyclase is sensitive to the C-terminal octapeptide of pancreozymin in all species investigated. 3. The enzyme is much more sensitive to secretin in rat and cat than in mouse and rabbit, whereas with guinea pig intermediate values are obtained. 4. The enzyme secretion is stimulated by secretin in pancreatic fragments of rat and cat, but not in those of mouse and rabbit. 5. These results suggest that in species where secretin stimulated enzyme secretion, it does so by stimulating the adenylate cyclase system.     

Biochemical aspects of the visual process. XXVIII. Classification of sulfhydryl groups in phodopsin and other photoreceptor membrane proteins.

Reaction of isolated bovine rod outer segment membrane with radioactive N-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule. On the basis of their reactivity towards rho-chloromercuribenzoate and rho-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with the rho-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and isprobably located in the vicinity of the chromophoric center. The differences between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) or N-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of the rho-chloromercuri-derivatives.     

在核苷酸三磷酸酶活化过程中核苷酸与Na~+之间的关系

<正> Na~++K~+-ATP酶反应序列包括依赖Na~+的磷酸化作用及依赖K~+的去磷酸化作用。具体包括五部分反应:1.Na~+和ATP与酶的高亲和结合部位任意结合;2.依赖Na~+和Mg~(++)的磷酸化反应,此步被ADP所抑制;3.从对ADP敏感的E_1～P转化成对K~+敏感的E_2-P;4.在K~+刺激下使E_2-P水解,释放无机磷;5.核苷酸促使E_2K→E_1K转换。 为探讨膜结合Na~+和K~+激活的核苷酸三磷酸酶活性及依赖Na~+的磷酸化反应的核苷酸特异性,我们选用ATP、CTP、ITP和GTP四种核苷酸做底物,观察Na~+与核苷酸的关系。     

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport

The Na⁺/K⁺-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na⁺ for extracellular K⁺ to the hydrolysis of ATP. The asymmetric distribution of Na⁺ and K⁺ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers–Post model. It involves the presence of gates alternatively exposing Na⁺/K⁺-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K⁺, information is lacking about Na⁺-occluded intermediates, as occluded Na⁺ was only detected in states incapable of performing a catalytic cycle, including two Na⁺-containing crystallographic structures. The current knowledge is that intracellular Na⁺ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na⁺-occluded intermediates, we isolated species with tightly bound Na⁺ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na⁺ becomes spontaneously occluded in the E1 dephosphorylated form of the Na⁺/K⁺-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na⁺ occlusion as it would have been expected; on the contrary, occluded Na⁺ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers–Post model for explaining the Na⁺ transport pathway.     

Purification and properties of pyrophosphatase of Acinetobacter johnsonii 210A and its involvement in the degradation of polyphosphate

Inorganic pyrophosphatase (E.C. 3.6.1.1) of Acinetobacter johnsonii210A was purified 200-fold to apparent homogeneity. The enzyme catalyzedthe hydrolysis of inorganic pyrophosphate and triphosphate to orthophosphate.No activity was observed with other polyphosphates and a wide variety oforganic phosphate esters. The molecular mass of the enzyme was estimatedto be 141 kDa by gelfiltration. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis indicated a subunit composition of six identical polypeptideswith a molecular mass of 23 kDa. The cation Mg² was required foractivity, the activity with Mn², Co² and Zn² was 48, 48 and 182% of the activity observed with Mg², respectively. The enzyme was heat-stable and inhibited by fluoride and iodoacetamide. The analysis of the kinetic properties of the enzyme revealed an apparent K_m for pyrophosphate of 0.26 mM. In A. johnsonii 210A, pyrophosphatase may be involved in the degradation of high-molecular polyphosphates under anaerobic conditions: (i) it catalyses the further hydrolysis of pyrophosphate and triphosphate formed from high-molecular weight polyphosphates by the action of exopolyphosphatase, and (ii) it abolishes the inhibition of polyphosphate: AMP phosphotransferase-mediated degradation by pyrophosphate and triphosphate.