Benjamin Kaminer

Identification and purification of a phenothiazine binding fragment from bovine brain calmodulin

Calmodulin is a small, acidic, intracellular calciumbinding protein which has been shown to influence the activity of a number of enzymic and structural systems in a calcium dependent manner (review [ 11). The protein, which is apparently ubiquitous in eukaryotes, has been implicated as one of the principal intracellular targets for the calcium released intracellularly following various external stimuli. Of the various probes used to study the role of calmodulin one of the more useful has been the class of antipsychotic drugs the phenothiazines which are able to bind to calmodulin in the presence of calcium, and block its modulating activity [2]. The specificity of the interaction of the various phenothiazines with calmodulin and the pharmacological significance of the interaction have been the subject of debate [3-S]. eluting from the column at -0.3-0.35 M NaCl in_ 20 mM Tris-HCl, 15 mM 2-mercaptoethanol, 1 mM EDTA (pH 7.8) was adjusted to a total concentration of 2 mM CaCla by addition of 1 /Sooth vol. 1 M CaClz to give -1 mM free Ca’+ion. The pool was then applied directly to a column (2.5 X 2dcm) of fluphenazineSepharose (prepared as in [7]) equilibrated in 50 mM imidazole, 200 mM NaCl, 1 mM CaCl,, 15 mM 2-mercaptoethanol (pH 7.2). Unbound material was washed from the column with 500 ml of this buffer, and the calmodulin then eluted with 300 ml of the same buffer but containing 5 mM EGTA in place of CaC12. Fractions (9 ml) were collected and the eluate monitored for calmodulin by alkaline urea slab-gel electrophoresis of samples from every tube. The calmodulin containing eluate was pooled, dialyzed against 2 changes of 50 vol. each of 2 mM Tris-HCl 15 mM 2mercaptoethanol (pH 7.8) and lyophilized. Here we have attempted to identify sites of interaction of phenothiazines on calmodulin using affinity chromatography of cyanogen bromide fragments of the molecule on fluphenazine-Sepharose.

DEUTERIUM OXIDE: INHIBITION OF CALCIUM RELEASE IN MUSCLE.

Calcium release, measured as luminescence of the protein aequorin, was measured simultaneously with membrane potential and isometric tension in single muscle fibers of the barnacle (Balanus nubilus). Deuterium oxide inhibited calcium release and isometric tension but did not affect membrane potential, a result consistent with the postulate that deuterium oxide inhibits the coupling between excitation and contraction.

The Role of Calcium on the Activity of ERcalcistorin/Protein-disulfide Isomerase and the Significance of the C-terminal and Its Calcium Binding A COMPARISON WITH MAMMALIAN PROTEIN-DISULFIDE ISOMERASE

ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI) shows a 55% identity with mammalian protein-disulfide isomerase (PDI) (Lucero, H. A., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112–23119) is a high capacity low affinity Ca2+-binding protein and behaves as a Ca2+ storage protein in the ER of a living cell (Lucero, H. A., Lebeche, D., and Kaminer, B. (1998) J. Biol. Chem. 273, 9857–9863). Here we show that recombinant ECaSt/PDI bound 26 mol of Ca2+/mol and a C-terminal truncated mutant bound 14 mol of Ca2+/mol, both with aK d of 2.8 mm in 50 mm KCl and 5.2 mm in 150 mm KCl. The percentage reduction in Ca2+ binding in the mutant corresponded with the percentage reduction of deleted pairs of acidic residues, postulated low affinity Ca2+-binding sites. 5 mm Ca2+ moderately increased the PDI activity of both ECaSt/PDI and the C-terminal truncated mutant on reduced RNase and insulin. Surprisingly, ECaSt/PDI in the absence of Ca2+prevented the spontaneous reactivation of reduced bovine pancreatic trypsin inhibitor. In the presence of 1–5 mmCa2+ (or 10 μm polylysine) ECaSt/PDI augmented the bovine pancreatic trypsin inhibitor reactivation rate. In contrast, the C-terminal truncated ECaSt/PDI augmented rBPTI reactivation in the absence of Ca2+ and 1–5 mmCa2+ further accelerated the reactivation rate, responses similar to those obtained with mammalian PDI.

Differentiation of a calsequestrin-containing endoplasmic reticulum during sea urchin oogenesis

We have used light and electron microscopic immunolocalization to study the distribution of a sea urchin calsequestrin-like protein (SCS) during sea urchin oogenesis. SCS was localized exclusively in the lumen of the endoplasmic reticulum (ER) and in the nuclear envelope of oocytes of all maturation stages. Immunoelectron microscopy also revealed that SCS is not present in golgi complexes of oocytes. Double label immunofluorescent staining of frozen sections of ovary with the SCS antiserum and an antibody to the cortical granule protein hyalin indicated a dramatic morphogenesis of the SCS-containing ER (SCS-ER) coincident with oocyte maturation. This differentiation included an apparent increase in the amount and complexity of the cytoplasmic SCS-ER network, the transient appearance of stacks of SCS-ER cisternae in synthetically active vitellogenic oocytes, and the restructuring of the SCS-ER in the cortex. Immunofluorescence of isolated oocyte cortices showed a plasma membrane-associated SCS-ER which was much less dense and regular than that found surrounding the cortical granules in the mature unfertilized egg cortex. Cytoplasmic and cortical microtubule arrays are present in oocytes and may provide the basis for the SCS-ER distributional dynamics. The results of this study underscore the dynamic nature of ER and how its organization reflects cellular functions. We suggest that the establishment during oogenesis of the dense SCS-ER tubuloreticulum provides the egg with the calcium sequestration and release apparatus that regulates calcium fluxes during egg activation and early development.

Hybrid myosin filaments from smooth and striated muscle.

Reconstituted myosin filaments from vertebrate smooth muscle are shorter than those from striated muscle ( Hanson & Lowy, 1964 ; Kaminer, 1969 ). In this investigation filaments were made from mixtures of the two types of soluble myosin in varying proportions. Mixtures were made of chicken gizzard myosin and either rabbit skeletal or chicken breast myosin. The filaments were made by rapid dilution of the KCl concentration to 0·1 m at pH 6·5. Negative staining was used for electron microscopy. Histograms showed no bimodal distribution of lengths. Thus the two types of myosin co-assemble to form “hybrid” filaments presumably through the association of complementary specific bonds in the two types of myosin. These “hybrid” filaments showed a progressive decrease of length with increasing proportions of smooth muscle myosin. Hence the smooth muscle myosin in some ways limits the length of the “hybrid” filaments. This limitation is observed when smooth muscle myosin constitutes as little as 5% of the mixture. Possible factors are discussed on the limitation of length of filaments including the possibility that smooth muscle myosin may have less specific bonds available for assembly.

CALMODULIN-ACTIVATED PHOSPHODIESTERASE FROM VERTEBRATE SMOOTH MUSCLE*

The potential roles of calmodulin in vertebrate smooth muscle, as in other tissues, are evidently diverse. Our previous studies have shown that calmodulin constitutes about 0.4% of the total protein of chicken gizzard,’ this represents a calcium-sensitive “switching” mechanism of considerable capacity. One of the potential functions of calmodulin in smooth muscle is to regulate cyclic nucleotide levels by altering phosphodiesterase activity. Cyclic nucleotides may influence smooth muscle contraction’ and the mechanisms controlling their levels may therefore be of considerable importance in the regulation of smooth muscle activity. The cyclic nucleotide phosphodiesterases of vertebrate smooth muscle may be separated from one another to some extent, and from calmodulin by DEAEcellulose chromatography of tissue extract^.^.^ In the presence of EDTA (20 mM Tris HCI. 1 mM EDTA, 15 mM 2-mercaptoethanol pH 7.8) the enzymes separate into two principal peaks of enzymic activity when eluted with a linear 0-0.5 M NaCl gradient in the same buffer. The peaks of enzyme activity elute at approximately 0.15 and 0.25 M NaCI. Acrylamide gel electrophoresis of eluate fractions show calmoddin .a elute at about 0.3 M NaCl. Only the first of the two peaks of enzymic activity is activated by addition of calmodulin in the presence of calcium. Nondenaturing alkaline acrylamide gels of samples from peak I enzyme show two bands when stained for phosphodiesterase activity. If the stain incubation is performed in the presence of calmodulin then the stain intensity of only one of the two bands is increased (FIGURE 1). This suggests that only this form of the enzyme is calmodulin activated. Gels of a comparable peak I enzyme obtained from a similiar preparation of bovine brain also showed two bands of activity only one of which is increased in intensity by incubation with calmodulin (FIGURE 1).The band corresponding to the calmodulin-activated phosphodiesterase has similar electrophoretic mobility in both the chicken gizzard and the bovine brain peak I enzyme fractions, however, the second band present in the peak I fractions from these tissues has considerably different mobility. If the peak I enzyme from the gizzard preparation is passed over an affinity column of calmodulin-Sepharose in the presence of calcium then a considerable proportion of the enzymic activity remains bound to the column. This enzyme may be eluted subsequently by application of an EGTA-containing buffer. The enzyme obtained in this way is activated by calmodulin in the presence of calcium and shows only a single band on acrylamide gels stained for phosphodiesterase activity (FIGURE 2). Thus at least three forms of phosphodiesterase have been identified in the gizzard, only one of which is activated by calmodulin. SDS gels of the affinity column purified enzyme show principally an 80,000