Judith Heldman

Cyclic AMP-dependent protein phosphorylation in chemosensory neurons: identification of cyclic nucleotide-regulated phosphoproteins in olfactory cilia.

Abstract Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine‐5′‐O‐(3‐thio)triphosphate (GTPγS). In control nonsensory (respiratory) cilia, the cyclic AMP‐dependent protein kinase is practically GTPγS‐insensitive. GTPγS activation of the olfactory enzyme appears to be mediated by a stimulatory GTP‐binding protein (G‐pro‐tein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [γ‐32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide‐dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 μM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP‐dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals.

Platelet activation and wheat germ agglutinin-binding

Platelet membrane contains a few glycoproteins, which are important to the aggregation phenomena [ 1,2]. Platelets interact with wheat germ agglutinin (WGA), possibly through glycoprotein I [3-51, and are agglutinated by this lectin. A glycoprotein extract, isolated from human platelet plasma membrane, exhibits haemagglutination and, in addition, causes agglutination of platelets [6]. We have postulated two possible mechanisms to explain this activity: (1) A lectin is buried in the membrane which, upon activation, becomes exposed. (2) Both lectin and the receptor may be present at the surface of the cell, but are self-neutralized in the freely-circulating platelet. Once the cell is activated, the pair-complex dissociates and intercellular interaction occurs outside the plane of the surface membrane. To further examine the availability of WGA receptors on the platelet membrane, and their possible exposure due to activation, we studied the binding of WGA to platelets before and during aggregation. We demonstrate here that there is no change in the binding of 1251-labeled WGA to washed human platelets, as a result of thrombin or ADP-induced aggregation. On the contrary, a marked decrease in the lectin binding was observed when the aggregation was performed in platelet-rich plasma (PRP).

Azidofluorescein diacetate — A novel intracellular photolabelling reagent

Many colloidal properties of the cell, such as those essential to sol-gel transformation, intracellular motion (cyclosis), ameboid movement, spindle formation and cell cleavage, depend for the most part, on the cytoplasmic matrix. Furthermore, the cytoplasmic matrix is the site of many fibrillar differentiations such as keratin fibers, myofibrils, microtubules and filaments. The viscosity of the cytoplasm is probably dependent on environment and internal factors such as the state of the microfilaments and microtubules. Therefore, it is of utmost importance and interest to label the cell cytoplasm and monitor molecular processes through the microviscosity changes. The viscosity of the membrane lipids [l-3] and the rotational relaxation time of lectins bound to the cellular surface membrane [4] have been measured using fluorescent probes. They were used also to study the lateral movement of membrane proteins [5,6] and the vertical displacement of membrane proteins [7]. In [8] the lipid soluble fluorescent probe 1,6diphenyl-1,3,5-hexatriene (DPH) was used to study membrane microviscosity change during platelet activation. Changes of the intracellular microviscosity of malignant and normal cells were studied by the introduction of fluorescein into the cell [9,10]. These studies suffer from the disadvantage that the free fluorescein leaks out of the cell, especially ai 37°C. We report here the introduction of an intracellular photoreactive labelling probe. This probe, azidofluorescein diacetate, is trapped inside the cell by rapid hydrolysis of the ester groups then is activated by irradiation. Using this method the fluorescein deriva-

Measurement of the intracellular pH of blood platelets using a photolabel fluorescent probe

The internal pH of blood platelets using the intracellular photolabel probe azidofluorescein diacetate was determined. No change of intracellular pH during thrombin activation of human platelets was observed. Platelets were found to adjust themselves very quickly to the external pH. Quantitative subcellular localization of the attachment sites of this probe reveals that most of it is bound to low molecular weight proteins or peptides.