The effects of p-mercuribenzenesulfonate on purified spectrin and actin

The compound p-mercuribenzenefulfonate was found to affect the self-association behavior of both spectrin and actin. The reagent brings about the depolymerization of F-actin, as judged from the decrease in the fluorescence of an attached pyrene label, with a second-order rate constant an order of magnitude less than that for the disruption of isolated erythrocyte cytoskeletons. Therefore, it is unlikely that the depolymerization of actin is the rate-determining step in the mercurial-dependent disruption of the erythrocyte cytoskeleton. Low reagent concentrations caused an initial rapid dissociation of spectrin tetramers at a rate comparable with that of cytoskeleton disruption. Prolonged incubation, or higher reagent concentrations, resulted in subsequent aggregation of spectrin. The reagent also prevented the interaction between spectrin and actin, presumably through its depolymerization of actin and its effects on spectrin. The early event in the disruption of isolated erythrocyte cytoskeletons by p-mercuribenzenesulfonate thus appears to be the dissociation of spectrin oligomers. Subsequent depolymerization of actin brought about by the reagent then results in total disruption of the cytoskeleton.     

Carbon cycling in a loblolly pine plantation

Summary The carbon cycle of a loblolly pine plantation in North Carolina was examined during its 12th through 16th years from planting. Net primary production during the study period averaged 2056 g C m^-2 year^-1. With autotrophic respiration equal to 2068 g C, the calculated gross production was 4124 g C m^-2 year^-1. Heterotrophic respiration of 694 g C m^-2 year^-1 resulted in net ecosystem production of 1362 g C m^-2 year^-1. In carbon cycle comparisons between forest ecosystems, autotrophic respiration rates were found to be closely coupled to regional temperature.     

Synthetic peptides used to locate the alpha-bungarotoxin binding site and immunogenic regions on alpha subunits of the nicotinic acetylcholine receptor

Synthetic peptides corresponding to 57% of the sequence of alpha subunits of acetylcholine receptors from Torpedo californica electric organ and extending from the NH2 to the COOCH terminus have been synthesized. The alpha-bungarotoxin binding site on denatured alpha subunits was mapped within the sequence alpha 185-199 by assaying binding of 125I-alpha-bungarotoxin to slot blots of synthetic peptides. Further studies showed that residues in the sequence alpha 190-194, especially cysteines-alpha 192, 193, were critical for binding alpha-bungarotoxin. Reduction and alkylation studies suggested that these cysteines must be disulfide linked for alpha-bungarotoxin to bind. Binding sites for serum antibodies to native receptors or alpha subunits were mapped by indirect immunoprecipitation of 125I-peptides. Several antigenic sequences were identified, but a synthetic peptide corresponding to the main immunogenic region (which is highly conformation dependent) was not identified.     

Solubilization of native actin monomers from human erythrocyte membranes

Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I.     

Use of procainamide gels in the purification of human and horse serum cholinesterases

Two large-scale methods based primarily on the use of procainamide-Sepharose gels were developed for the purification of horse and human serum non-specific cholinesterases. With method I, the procainamide-Sepharose 4B gel was used in the first step to handle large volumes of serum. With method II, the procainamide-Sepharose 4B gel was used in the final step to obtain pure enzyme. Although both methods gave electrophoretically pure cholinesterase preparations in good yields, they were significantly more efficient at purifying the horse enzyme than the human enzyme. To study this problem, the relative binding of human and horse cholinesterases to procainamide-, methylacridinium (MAC)-, m-trimethylammoniophenyl (m-PTA)- and p-trimethylammoniophenyl (p-PTA)-Sepharose 4B gels were measured, by using two approaches. In one, binding was measured by a procedure involving equilibration of pure cholinesterase in a small volume of diluted gel slurry (4%, v/v). A partially purified preparation of Electrophorus acetylcholinesterase was included. Pure human cholinesterase bound consistently more tightly to each of the gels than did horse cholinesterase, and the acetylcholinesterase appeared to bind the gels 10-100 times more tightly than did the non-specific cholinesterases. The order of binding for the cholinesterases, beginning with the tightest, was: procainamide-Sepharose 4B, MAC-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. For the acetylcholinesterase the order was: MAC-Sepharose 4B, procainamide-Sepharose 4B, p-PTA-Sepharose 4B and m-PTA-Sepharose 4B. The second approach involved passing native sera or partially purified sera fractions through 1 ml test columns of each of the four affinity gels to determine their retention capacity for the cholinesterases. With these impure samples, the MAC-Sepharose 4B gels proved superior to the procainamide-Sepharose 4B gels at retaining human cholinesterase, but the opposite was true for the horse cholinesterase.     

Subcellular location of an abundant substrate (p36) for tyrosine-specific protein kinases.

A 36,000-dalton cellular protein (p36) has been identified previously as an abundant substrate for phosphorylation by tyrosine-specific protein kinases. Since several of the responsible kinases are associated with the plasma membrane, we explored the subcellular distribution of p36. Biochemical fractionations located p36 on the plasma membrane of both normal and retrovirus-transformed cells. Approximately half of the p36 was bound to the membrane with the affinity of a peripheral membrane protein; the remainder was even more tightly bound. The distribution of p36 among subcellular fractions and its affinity for the plasma membrane were not affected by tyrosine phosphorylation. We determined that p36 is synthesized in the soluble compartment of the cell and then moves rapidly to the membranous compartment. Immunofluorescence microscopy with antibodies directed against p36 revealed two distinct distributions of the antigen: (i) a sharply demarcated crenelated pattern within or immediately beneath the plasma membrane, which we presume to be a correlary of the distribution of p36 in biochemical fractionations; and (ii) diffuse staining in a cytoplasmic location that could not be attributed to a specific feature of cytoarchitecture and could not be easily reconciled with the results of biochemical fractionations. Efforts to detect the secretion of p36 were unsuccessful. No evidence was obtained for exposure of p36 on the cell surface, and no changes in localization were observed as a consequence of neoplastic transformation. During the course of this study, we had the opportunity to pursue a previous report that p36 is a component of the enzyme malate dehydrogenase (Rubsamen et al., Proc. Natl. Acad. Sci. U.S.A. 79:228-232, 1982). We were unable to substantiate this claim. We conclude that at least a substantial fraction of p36 is located on the cytoplasmic aspect of the plasma membrane, where it could be well situated to serve as a substrate for several identified tyrosine-specific kinases. But the function of p36 and its role, if any, in neoplastic transformation of cells by retroviruses possessing tyrosine-specific kinases remain enigmatic.     

Quantitative Analyses of Certain Enteric Bacteria and Bacterial Extracts: II. Discrimination of Sonic Extracts by Interfacial Densitometry of Precipitin Systems

Bacterial extracts prepared by ultrasonic disruption were reacted with both narrow- and broad-spectrum reference (homologous) and cross-reacting (heterologous) precipitins produced in rabbits. Quantitation of the reaction was obtained by densitometry of the antigen-antibody interface. Comparisons were made of sonic extracts from various starting populations all equated to the same nitrogen concentrations, and of various nitrogen levels derived from five bacterial population levels prepared separately. Sources of error are probed to show under what circumstances cross-reactions would be of greater magnitude than reference ones. The feasibility was shown of using quantitative densitometry of the interface combined with broadly reacting precipitins to identify bacteria on an intergeneric and interspecies scale. Problems associated with the use of absorbed or monospecific precipitins are explained.     

Age and growth of Lutjanus kasmira (Forskål) in Hawaiian waters

The growth of Hawaiian taape, Lutjanus kasmira , was studied by examining otoliths and by analysing length-frequency distribution. Annual hyaline and opaque markings were visible in whole mounts of sagittae, which were verified by enumeration of daily increments with a scanning electron microscope (SEM) and through marginal increment analysis. The von Bertalanffy growth curve was fitted to the data, resulting in: where t. l . is total length (cm) and t is age (years). SEM observations revealed that the slowgrowth hyaline zones were composed of daily increments too small (0.4–0.8 μm) to be resolved optically. Thus, age estimates derived by numerically integrating otolith growth rate data obtained with a light microscope showed a negative bias, resulting in overestimation of growth rates. Parameter estimates obtained from three different types of length-frequency analysis were also unstable. This was due, at least in part, to differences in the size composition of fish sampled with different fishing gears and from different depths.
The growth rate registered in Hawaii falls within the reported growth coefficients of lutjanids, whereas it is one of the highest in the Pacific and clearly higher than a deep-water lutjanid species growth in Hawaii. Probably, this high growth rate may have been enhanced by the relative lack of competitors in the depauperate Hawaiian marine fish community.