Epidermal growth factor receptor binding is not a simple one-step process

The binding kinetics of 125I-labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture were measured at 4 degrees C. Initial binding rates as a function of hormone concentration allowed estimation of simple two-state on-off rate constants of 1.2 x 10(6) M-1 s-1 and 4.9 x 10(-3) s-1, respectively. These two-state parameters gave inadequate computer fits to long term kinetic and equilibrium-binding data, suggesting that an additional process(es) was occurring. Nonlinear equilibrium Scatchard plots and transient "pseudo-Scatchard" plots taken at pre-equilibrium times support the idea that at least one other process is occurring during receptor binding. 125I-EGF-receptor dissociation kinetic plots were biphasic, yielding rate constants of 1.5 x 10(-2) s-1 and 5.6 x 10(-5) s-1 with the ratio of the two components changing with the time of initial incubation with 125I-EGF. Application of a ternary complex model which assumed complexation of the bound receptor with a cell surface interaction molecule gave satisfactory fits to all data.     

Use of an affinity label to probe the function of the NADPH binding component of the respiratory burst oxidase of human neutrophils

The respiratory burst oxidase of neutrophils can be activated in a cell-free system in which solubilized membranes, cytosol, and Mg2+ are required and in which sodium dodecyl sulfate is used to convert the dormant oxidase to an active form. The 2',3'-dialdehyde analog of NADPH was used as an affinity label for the cytosolic NADPH binding component of the respiratory burst oxidase from human neutrophils. When treated with this affinity label in the presence of sodium cyanoborohydride to reduce Schiff bases, neutrophil cytosol was shown to lose at least 90% of its activity in the cell-free system. In contrast to normal cytosol, treated cytosol had lost its ability to abolish the lag time required for activation of the oxidase, suggesting that the treated cytosol was no longer able to participate in the rate-limiting activation step. Furthermore, the treated cytosol had lost its ability to convert the oxidase from a form with a high Km to a form with a low Km for NADPH. The ability of dialdehyde-treated cytosol to activate the oxidase could be restored by untreated cytosol with a concentration dependence suggesting that only one kinetically active component of the oxidase was inhibited by treatment with the NADPH analog. Like the dialdehyde-treated cytosol, cytosols from patients with chronic granulomatous disease caused by a deficiency in a cytosolic Mr = 47,000 protein (pp47) fail to participate in the rate-limiting activation step (Curnutte, J. T., Scott, P. J., and Babior, B. M. (1989) J. Clin. Invest. 83, 1236-1240). These chronic granulomatous disease cytosols were nevertheless able to restore limited activity to the dialdehyde-inactivated cytosol in a cell-free activation system. These results are consistent with a model in which (a) the NADPH binding subunit of the oxidase exists in a very slowly dissociating complex with one or more additional cytosolic components, including pp47, and (b) the NADPH binding component of the oxidase controls the affinity of the enzyme for NADPH, either directly or through the binding of additional cytosolic factors.     

The use of high-spectral-resolution radiometer data for detection of low chlorophyll concentrations in Lake Kinneret

Chlorophyll distribution in Lake Kinneret was estimated in aperiod of low chlorophyll-a concentrations (3–7 mg m^–3)using remotely sensed data. The data set included high-spectral-resolutionradiometric measurements in the range 400–750 nm, chlorophylland suspended matter concentrations, Secchi disk transparencyand vertical attenuation coefficients at 20 stations. The spectroradiometricdata were used to create the algorithms suitable for quantitativedetermination of chlorophyll content. The present paper presentsexperimental field evidence showing that fluorescence can besuccessfully used for remote monitoring of chlorophyll-a content(with an estimation error <0.5 mg m^–3) in productiveinland waters with a background of variable and relatively highsuspended matter concentration.     

Modes of reproduction in Australian populations of Hypericum perforatum L. (St. John's wort) revealed by DNA fingerprinting and cytological methods.

Hypericum perforatum L. (St. John's wort) is widely used in homeopathic medicine, but has also become a serious weed in Australia and many other countries. Reproduction in H. perforatum was investigated using markers based on restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP). Between two Australian populations, plants displayed 14 polymorphisms from a total of 22 scorable RFLP markers when genomic DNA was probed with M13 bacteriophage, but individuals within each population exhibited identical RFLP fingerprints. Ninety-four percent of the progeny of four crosses made between the two populations exhibited identical fingerprint and ploidy level to the maternal parent, and probably originated apomictically. Seven seedlings with recombinant RFLP or AFLP fingerprints were found from a total of 121 progeny. Both molecular marker techniques detected the same recombinants from a subset of screened progeny. Cytological analysis showed that the seven recombinants comprised three tetraploids (2n = 4x = 32), three hexaploids (2n = 6x = 48), and one aneuploid (2n - 1 = 31), which suggested that the level of normal reduced embryo sacs was only 2.5%. These results are discussed in relation to the management of invasive populations, and the implications for plant breeding and production of St. John's wort for medicinal purposes.     

Divalent cation binding to reduced and octanoyl acyl-carrier protein

Mn(II) EPR binding studies with reduced acyl-carrier protein (ACP-SH) strongly suggest the presence of two relatively high-affinity manganese-binding sites (average Kd/site approximately 80 microM) at physiological pH. Lowering the pH or titrating with sodium chloride reduces the average number of bound divalent cations and decreases the binding affinity. This is consistent with the idea that anionic ligand(s), e.g. the carboxylate of glutamic or aspartic acid, on the protein are involved in manganese ion coordination. At pH values above 8.0, binding affinity is also reduced, whereas the average number of bound metal ions increases to about five at pH 8.5. By interacting weakly with divalent cations (average Kd/site approximately 1 mM), octanoyl acyl-carrier protein (OcoACP) exhibits dramatically different metal-ion-binding properties compared to ACP-SH. Calcium and magnesium can compete in either ACP species for manganese binding. Photochemically-induced dynamic nuclear polarisation 1H-NMR experiments strongly suggest that ACP-SH and OcoACP undergo at pH-induced conformational change between pH 5.5 and pH 7.0, and that divalent cations stabilize the protein against such pH-induced structural perturbations.     

Acid protease expression and regulation in the embryonic proventriculus of birds

Normal embryonic proventriculi and the heterospecific recombinants of proventricular endoderm and mesenchyme were transplanted onto the chorio-allantoic membrane, and electrophoretic patterns of acid proteases in the explants were analyzed. The results demonstrated that the 6-day chick and 5-day quail proventricular endoderm produces acid proteases according to its own genetic information even under the influence of heterospecific mesenchyme, and that the production of acid proteases is regulated by some humoral factors of the hosts.     

Anthocyanin pigments and leaf flavonoids in the family araceae

Anthocyanins, variously identified in inflorescence, fruit, leaf or petiole of 59 representative species of the Araccae, are of a simple type. The most common pigment is cyanidin 3-rutinoside, while pelargonidin 3-rutinoside and cyanidin 3-glucoside are regularly present. Two rare pigments are: cyanidin 3-gentiobioside in Anchomanes and Rhektophyllum, both in the subfamily Lasioideae; and delphinidin 3-rutinoside in Schismatoglottis concinna. In a leaf survey of 144 species from 58 genera, flavone C-glycosides (in 82%) and proanthocyanidins (in 35–45%) were found as the major flavonoids. In the subfamily Calloideae, subtribe Symplocarpeae, flavonols replace glycoflavones as the major leaf components but otherwise flavonols are uncommon in the family (in 27% of the sample) and more usually co-occur with flavone C-glycosides. Two new flavonol glycosides were characterized from Lysichiton camtschatcense: kaempferol 3-(6-arabinosylgalactoside)and kaempferol 3-xylosylgalactoside. Simple flavones, luteolin and chrysoeriol (in 6%) were found only in the subtribes Arinae and Cryptocoryninae, subfamily Aroideae. Flavonoid sulphates were identified in only four taxa: glycoflavone sulphates in two Culcasia species and Philodendron ornatum and a mixture of flavone and flavonol sulphates in Scindapsus pictus. Caffeic ester sulphates were more common and their presence in Anthurium hookeri was confirmed. These results show that the Araceae are unusual amongst the monocots in their simple and relatively uniform flavonoid profile; no one subfamily is clearly distinguished, although at tribal level some significant taxonomic patterns are observed. The best defined groups are the subfamilies Lasioideae and Monsteroideae, and the tribes Symplocarpeae and Arophyteae, and the subtribe Arinae. The greatest chemical diversity occurs in Anthurium and Philodendron, but this may only reflect the fact that these are the two largest genera in the family. The origin and relationship of the Araccae to other monocot groups are discussed in the light of the flavonoid evidence.     

Quantitation of submandibular proteins resolved from normal individuals and children with cystic fibrosis

Submandibular secretions collected from children with cystic fibrosis (CF) showed increased protein concentration (milligrams/milliliter) and increased amylase specific activity (units/milligram of protein) relative to normal secretions. These differences between normal (N) and CF secretions were as follows: protein, 1.25 ± 0.51 (N), 1.75 ± 0.35 (CF) (P < 0.02); and amylase, 58 ± 18 (N), 80 ± 19 (CF) (P < 0.001). To determine the basis for elevated protein in CF saliva, several major proteins resolved by polyacrylamide disc gel electrophoresis were quantitated by densitometry. These included four phosphoproteins (PP), serum albumin, an acid phosphatase-containing fraction, amylase, and an unidentified protein referred to as PI-7.1. Together, these proteins comprise greater than 75% of the total protein in the secretion. Differences in individual protein concentrations (milligrams/milliliter) resolved from normal and CF secretions, respectively, were as follows: PP₂, 0.02 ± 0.01, 0.03 ± 0.02 (NS, not significant); PP₃, 0.06 ± 0.04, 0.05 ± 0.03 (NS); acid phosphatase fraction, 0.06 ± 0.04, 0.12 ± 0.07 (P < 0.05); amylase, 0.09 ± 0.04, 0.27 ± 0.16 (P < 0.01); and pI-7.1, 0.04 ± 0.02, 0.13 ± 0.08 (P < 0.02). Amylase, the most significant contributor to the elevated protein, comprised 26% of the total protein of normal secretions and 38% of the total protein of CF secretions. Thus, our results do not support the concept of a generalized increase in all organic components in CF submandibular secretions but, rather, increases in specific proteins, namely amylase, component pI-7.1, and an acid phosphatase-containing fraction.