Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.     

Biochemical characterization of a family of serine/threonine protein kinases regulated by tyrosine and serine/threonine phosphorylations.

Mitogen-activated protein kinase (p42mapk) becomes transiently activated after treatment of serum-starved murine Swiss 3T3 cells or EL4 thymocytes with a diversity of mitogens. Similarly, a meiosis-activated protein kinase (p44mpk) becomes stimulated during maturation of sea star oocytes induced by 1-methyladenine. Both p42mapk and p44mpk have been identified as protein-serine/threonine kinases that are activated as a consequence of their phosphorylation. Because homologous protein kinases may play essential roles in both mitogenesis and oogenesis, we have compared in detail the biochemical properties of these two kinases. We find that these kinases are highly related based on their in vitro substrate specificities, sensitivity to inhibitors, and immunological cross-reactivity. However, they differ in apparent molecular weight and can be separated chromatographically, indicating that the two enzymes are distinct. Furthermore, in the course of this investigation, we have identified a 44-kDa protein kinase in mitogen-stimulated Swiss mouse 3T3 cells and EL4 thymocytes that co-purifies with p44mpk and thus appears to be a closer homolog of the sea star enzyme. Analysis of these protein kinases clarifies the relationships between a set of tyrosine-phosphorylated 41-45-kDa proteins present in mitogen-stimulated cells (Martinez, R., Nakamura., K. D., and Weber, M. J. (1982) Mol. Cell. Biol. 2, 653-655; Cooper, J. A., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37), two myelin basic protein kinases identified in epidermal growth factor-treated Swiss mouse 3T3 cells (Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G. (1990) J. Biol. Chem. 265, 11487-11494), and p42mapk. Our work points to the existence of a group of related serine/threonine protein kinases, regulated by tyrosine phosphorylation and functioning at different stages of the cell cycle.     

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.     

c-mos proto-oncogene product is partly degraded after release from meiotic arrest and persists during interphase in mouse zygotes.

Recently, it has been shown that the product of the c-mos proto-oncogene is a component of cytostatic factor, an activity present in unfertilized eggs from vertebrates that arrests the cell cycle in metaphase of the second meiotic division (metaphase II) possibly by stabilizing maturation-promoting factor (MPF). We have studied the behavior of the c-mos product in metaphase II mouse oocytes and soon after activation. The amount of c-mos in the oocyte was still very high after second polar body extrusion, when cyclin B has been degraded and MPF activity had decreased dramatically. Degradation of c-mos takes place later, during the G1 phase of the first cell cycle and a residual amount of c-mos is detectable during the first zygotic interphase. Our data show that the degradation of c-mos is not involved in the release from the metaphase arrest.     

The spatial distribution of cations in nematocytes of Hydra vulgaris

The spatial distribution of cations was assayed qualitatively and quantitatively in tentacular nematocytes of Hydra vulgaris in a scanning transmission electron microscope by means of x-ray microanalysis performed on 100 nm thick freeze-dried cryosections. The matrix of undischarged cysts (stenoteles, desmonemes and isorhizas) was found to contain mainly K⁺. In isolated nematocysts of Hydra the intracapsular potassium can be readily substituted by practically any other mono- and divalent cation (Na⁺, NH₄ ⁺, Mn²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺) all, except Fe²⁺, without impairing the ability of the cyst to respond to the chemical triggering with dithioerythritol or proteases. Monovalent cations increase the osmotically generated intracapsular pressure compared to divalent ions.     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane—isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3–300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been succesfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at −20°C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Class II tubulin, the major brain beta tubulin isotype is polyglutamylated on glutamic acid residue 435.

Protein sequencing shows that porcine brain tubulin retains the N-terminal sequences of alpha and beta tubulin after a mild treatment with subtilisin. C-terminal peptides released by subtilisin were purified and characterized by automated Edman degradation and mass spectrometry. We confirm the polyglutamylation of alpha tubulin on glutamic acid residue 445 reported by others and show in addition that class II beta tubulin, the major beta tubulin isotype of adult brain, is also polyglutamylated. The substitution is restricted to glutamic acid residue 435. Thus all major tubulin isotypes of adult brain are subjected to polyglutamylation.     

How Small Are the Smallest Selectable Domains of Form?

Two lines of Drosophila melanogaster from the same base population were selected in opposite directions to produce simultaneous antagonistic changes in two very small (less than 0.2 mm) and closely adjacent (less than 0.3 mm) dimensions within the base of the wing. Wing dimensions near the targeted area became differentiated by large positive and negative percentage differences, while only small homogeneous percentage changes occurred in the remainder of the wing. If very small regions of morphology (less than 100 cells across) can respond to selection almost independently, even in small population samples, then the control of developmental detail must involve many genes, and the diversity of possible outcomes in development and adaptation must be large.     

A metal ion-binding site in the kringle region of bovine prothrombin fragment 1.

45Ca(II) binding studies (equilibrium dialysis) on the kringle domain of bovine prothrombin fragment 1 were conducted using a mixture of peptides (residues 43-156 and 46-156) resulting from limited alpha-chymotryptic hydrolysis of fragment 1. Analysis of the Scatchard plot of these data indicates a single, low affinity Ca(II)-binding site to be present. Similar results were obtained from studies on the decarboxylated fragment 1 derivative, 10-gamma-MGlu-fragment 1. Acetylation of bovine fragment 1 in the absence of Ca(II) or Mg(II) ions results in the loss of the metal ion-promoted quenching of the intrinsic Trp fluorescence of the protein and the Ca(II)-mediated binding to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles. The acetylation of the NH2 alpha-group of Ala-1 has been shown (Welsch, D. J., and Nelsestuen, G. L. (1988) Biochemistry 27, 4946-4952) to abolish the PS/PC binding property of fragment 1. The present study demonstrates that acetylation of a second site possibly Ser-79 or Thr-81 using the conditions described in the preceding paper results in loss of both the fluorescence transition and the Ca(II)-mediated PS/PC binding of the resulting protein derivative. Removal of the O-acetyl group at the Ser-79/Thr-81 site is accomplished by aminolysis with 0.2 M hydroxylamine, pH 10, 50 degrees C; the fluorescence transition is partially restored. PS/PC binding is partially restored if the NH2 alpha-group of Ala-1 is trinitrophenylated but is not restored if the NH2 alpha-group of Ala-1 is acetylated. We conclude that the Ser-79/Thr-81 site may represent a portion of the metal ion-binding site within the kringle domain of fragment 1. Occupancy of this site by a Ca(II) ion appears to be important in the binding of the protein to PS/PC vesicles.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.