Irreversible degradation of histidine-96 of prothrombin fragment 1 during protein acetylation: another unusually reactive site in the kringle

Acetylation of prothrombin fragment 1 in acetate-borate buffer at pH 8.5 resulted in the appearance of increased light absorbance at about 250 nm. Protease digestions resulted in isolation of a single peptide (residues 94-99) with intense absorbance at about 250 nm (estimated extinction coefficient of 5000 M-1 cm-1). Amino acid analysis showed the expected composition except for the absence of His-96. Instead, an unidentified amino acid which had a ninhydrin product with absorption properties similar to those of proline eluted near aspartate. When sequenced, this peptide (YP?KPE containing epsilon-amino-acetyllysine) lacked histidine at the third position but gave a high yield of a PTH derivative that eluted near PTH-Gly from the HPLC column. Fast atom bombardment mass spectrometry of the derivatized 94-99 peptide showed a mass that was 74 units higher than expected. The histidine degradation product was identified as a di-N-acetylated side chain with an opened imidazole ring and loss of C2 of the ring. While a similar degradation pattern has previously been reported during acylation of histidine, the high chemical reactivity exhibited by His-96 was unusual. For example, under conditions sufficient for quantitative derivatization of His-96, His-105 of fragment 1 was not derivatized to a detectable level. Furthermore, His-96 in fragment 1 was at least an order of magnitude more susceptible to degradation than His-96 in the isolated 94-99 peptide. His-96 is therefore one of several neighboring amino acids of the kringle portion of fragment 1 that displays highly unusual chemistry (see also Asn-101 [Welsch, D.J., & Nelsestuen, G. L. (1988) Biochemistry 27 4946-4952] and Lys-97 [Pollock, J.S., Zapata, G.A., Weber, D.J., Berkowitz, P., Deerfield, D.W., II, Olson, D.L., Koehler, K.A., Pedersen, L.G., & Hiskey, R.G. (1988) in Current Advances in Vitamin K Research (Suttie, J.W., Ed.) pp 325-334, Elsevier Science, New York]).(ABSTRACT TRUNCATED AT 250 WORDS)     

Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1

The binding of Tb3+ to bovine prothrombin and the amino-terminal 156 residues of prothrombin (F-1) was studied. On the basis of various Tb3+ emission properties, three classes of Tb3+-binding sites were described. The first class contained three high affinity sites in the F-1 region. These sites were filled noncooperatively and were saturated with Tb3+ before the other classes of sites started to fill. Ho3+ quenching of Tb3+ emission showed that these sites were in close proximity to one another (estimated distances 6-12 A). The second class of sites contained three lower affinity sites, also in the F-1 region. These sites bound Tb3+ in a stoichiometric manner and saturated prior to metal binding to the final class of sites. The number of protein ligands binding Tb3+ in the high affinity sites decreased as this second set of sites was filled. Ho3+ quenching of Tb3+ emission suggested that these sites were closely spaced and/or close to the first set of sites. The third class of sites contained 4-6 low affinity sites unique to prothrombin (not in the F-1 region). These sites were not studied extensively, but Tb3+ did not appear to bind stoichiometrically and did not saturate these sites in a manner similar to the other two classes of sites. The emission properties of Tb3+ bound to F-1 were different in KCl versus NaCl containing buffer while the emission properties of Tb3+ bound to prothrombin were not. Optimum conditions for studying lanthanide binding to F-1 (i.e. when Tb3+ bound to F-1 showed emission properties similar to Tb3+ bound to prothrombin) were when F-1 experiments were done at low F-1 concentrations in buffer containing 0.1 M KCl.     

Electron microscopy and hydrodynamic properties of blood clotting factor V and activation fragments of factor V with phospholipid vesicles

The electron microscopic and hydrodynamic properties of factor V and factor Va-vesicle complexes were determined. Images of negatively stained factor V bound to vesicles showed the protein as a relatively large globular domain (9.5 nm diameter) connected to the membrane through a narrow protein region 0.5-3 nm in length. This connecting region was not always visible and was measured as the distance between the globular region and the apparent vesicle edge. Factor V protein alone usually appeared as two connected globular regions of 10.2 and 6.5 nm diameter. The two-domain protein structure appeared consistent with both the image of factor V alone and bound to the membrane. Factor V had no biological activity in a phospholipid-free prothrombinase assay system used. The proteolytically activated form of factor V generated by digestion with thrombin (factor Va) was at least 30,000 times more active. The electron microscopic images of factor Va-vesicle complexes showed a smaller protein that was more closely associated with the vesicle surface than was factor V. The light chain (Mr about 80,000) component of factor Va also bound to the surface of the vesicles and appeared to be largely external to the membrane. Protein-induced hydrodynamic radius changes for the factor V-vesicle and factor Va-vesicle complexes were 12.8 and 6.3 nm, respectively. The images observed in the electron microscope were used to calculate protein-induced radius changes. Comparison of these values with the experimentally determined hydrodynamic radius changes showed approximate agreement for factor Va-membrane complexes. However, the images of factor V-vesicle complexes suggested smaller hydrodynamic radius changes than were actually observed.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

H－ras在颊粘膜正常、良性、癌前及恶性变上皮中的表达：免疫组织化学定量研究

本文通过建立图象分析方法对免疫组织化学反应结果进行定量,检测观察Ｈ－ｒａｓ在口腔颊粘膜上皮在正常（Ｎ）、慢性炎症（ＩＦ）、癌旁上皮（ＥＡＣ）和鳞癌（ＳＣＣ）的变化过程中的表达并进行分析。结果显示Ｈ－ｒａｓ在ＳＣＣ组中,以中等分化的ＳＣＣ无论是Ｈ－ｒａｓ表达的量还是细胞阳性率都较高。此外,组织学观察显示,Ｈ－ｒａｓ在处于分化末期但尚未角化的正常上皮细胞中有较高的表达。本文结果显示了Ｈ－ｒａｓ的过表达与上皮细胞的会化程度密切相关。本研究还显示,所采用的阳性区域透光值、平均总透光值及阳性反应区域与阴性反应区域比值可靠并有相关性。这进一步说明了用免疫组化定量方法检测Ｈ－ｒａｓ癌基因表达的精确和可靠性。     

Prothrombin fragment 1-membrane interactions: a calcium-43 NMR study

The 43Ca NMR spectra are reported for solutions of prothrombin fragment 1 in the presence and absence of phospholipid. The calcium NMR spectrum permits distinction between three thermodynamic classes of calcium-binding interactions. The calcium ion in the lipid-free solutions was labile, with maximum residence times estimated for the average protein site in the range of 0.5-1 ms. The calcium spectrum was sensitive to the protein association and the addition of phospholipid, which appears to sharpen the calcium specificity for the protein sites. The calcium NMR spectra in the presence of phospholipid are similar to those in lipid-free solutions, which suggests that the calcium ion remains labile in the lipid-protein complex.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Lectin-binding sites on the plasma membranes of rabbit spermatozoa: Changes in surface receptors during epididymal maturation and after ejaculation

Modifications in rabbit sperm plasma membranes during epididymal passage and after ejaculation were investigated by used of three lectins: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.     

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins that bind calcium in a phospholipid-dependent manner

Three proteins (Mr = 64K, 32K, and 22K) that bind to phospholipids in a calcium-dependent manner were purified from bovine brain. The calcium-binding properties of these proteins were investigated by equilibrium dialysis and by gel filtration chromatography. The 64- and 32-kDa proteins were found to have calcium- and phospholipid-binding properties strikingly similar to those of protein kinase C [Bazzi, M.D., & Nelsestuen, G.L. (1990) Biochemistry 29, 7624]. The free proteins bound limited divalent metal ion even at 200 microM calcium. However, they bound eight to nine calcium ions per protein in the presence of membranes containing acidic phospholipids. The calcium concentrations needed for protein-phospholipid binding were different for these two proteins and were strongly influenced by the phospholipid composition of the vesicles; vesicles of higher phosphatidylserine content required lower concentrations of calcium for protein-membrane association. These properties described a general type of calcium-interacting system where simultaneous interaction of all three components (protein, phospholipids, and calcium) is required. The free proteins may provide only partial coordinate bonds to each calcium ion, but complete calcium-binding sites could be generated at the protein-phospholipid interface. In contrast to the 64- and 32-kDa proteins, the 22-kDa protein bound similar amounts of calcium (two to three ions/protein) in the presence or the absence of phospholipids. The 22-kDa protein had the lowest affinity for phospholipid and the highest affinity for calcium of the three proteins tested. Thus, calcium-dependent phospholipid-binding proteins consist of several types. For example, the 64- and 32-kDa proteins appear to be quite abundant and may even function as a calcium buffer to modulate signaling events.