Isolation and characterization of three forms of 36-kDa Ca2+-dependent actin- and phospholipid-binding proteins from human placenta membrane

We purified three forms of 36-kDa proteins, two monomeric 36-kDa proteins, which had pIs of 7.5 (36K-I) and 6.4 (36K-II), and one 36-kDa complex (36K-C) consisting of two subunits, 36-kDa (pI 7.5) and 12-kDa (pI 5.8), from human placenta membrane. The 36-kDa subunit of 36K-C was identical to 36K-I as judged by pI, cyanogen bromide peptide mapping and immunological cross-reactivity. The three proteins showed F-actin- and phosphatidylserine-binding abilities dependent on Ca2+ concentrations at millimolar and micromolar levels, respectively. They all had phospholipase A2 inhibitory activity. Only 36K-II was phosphorylated extensively at tyrosine residue in Ca2+- and EGF- dependent manners in the membrane fraction of A431 cells. 36K-I was the best substrate for src kinase, whereas 36K-II was the best for fps kinase. However, 36K-C was not phosphorylated by any kinases used here.     

Characterizations of two distinct Ca2+-dependent phospholipid-binding proteins of 68-kDa isolated from human placenta

Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.     

Twenty-eight-kilodalton phosphorylatable calcium- and lipid-binding proteins purified from Physarum plasmodium.

Two 28-kDa calcium- and lipid-binding proteins were isolated from a detergent-insoluble fraction of the Physarum plasmodium. Both proteins have molecular masses of approximately 28 kDa by SDS-PAGE. The protein designated 28K-I has a slightly lower mobility than that designated 28K-II. The purified 28K-I has a dissociation constant of 1.0 microM for Ca2+ ions, while the 28K-II has two different dissociation constants: one of 0.32 microM and the other of 3.2 mM. The 28K-I binds to liposomes at Ca2+ concentrations higher than 1.0 microM and has a dissociation constant for lipid of 34 micrograms/ml at 10 microM Ca2+. The 28K-II binds to liposomes at concentrations of Ca2+ above the mM range and has a dissociation constant of 36 micrograms/ml for lipid at 2 mM Ca2+. There is no evidence of actin-binding activity by either of the 28-kDa (28K) proteins. The 28K proteins crossreacted with an antiserum against chicken brush border calpactin I. The two proteins have quite different phosphorylation levels between a fraction prepared from the cytosolic endoplasm and a fraction prepared from the whole cell. The 28K proteins may play some role in the membrane structure dynamics of the cortical gel layer.     

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2.

The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.     

Purification, characterization, and partial sequence analysis of 32-kDa calcimedin from chicken gizzard

Calcimedin is a group of proteins which has a binding ability to several hydrophobic matrices or cellular membrane fractions in the presence of Ca2+. Although the molecular properties were partially clarified, the physiological functions of calcimedins have not been clearly defined. In this study, we describe the isolation and characterization of 32-kDa calcimedin from chicken gizzard. Both structural and functional studies establish that 32-kDa calcimedin is a member of the calpactin/lipocortin family. The 32-kDa calcimedin displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding activity, and phospholipid binding activity similar to those of calpactins/lipocortins. Antiendonexin II antibody recognized 32-kDa calcimedin. However, antibodies against calpactin I (lipocortin II), calpactin II (lipocortin I), 35-kDa calcimedin, and 67-kDa calcimedin did not cross-react with 32-kDa calcimedin. One-dimensional peptide maps of the 32-kDa calcimedin and the 35-kDa calcimedin are different, confirming that they are distinct proteins. By comparing the sequence of 32-kDa calcimedin with the predicted sequence of endonexin II, we concluded that the primary structure of the 32-kDa protein is highly conserved. In particular, the sequences AMKGMGTDDEXEIXL, GMGTDEEEIL, VLTEILASR, and ILTSR conform to the endonexin consensus sequence, which is characteristic of the calpactin/lipocortin family.     

The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells

Human monoblast U937 cells contain a soluble phospholipase A2 (PLA2) that is activated over the range of 150-600 nM Ca2+ and is stable only at neutral pH. We have purified this PLA2 over 34,000-fold to near homogeneity using sequential ion exchange, hydrophobic interaction, and gel filtration chromatography steps. The protein has a Mr of approximately 100,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and an isoelectric point of 5.1. Four lines of evidence indicate that this 100-kDa polypeptide represents the PLA2. (i) The intensity of staining of the 100-kDa protein was proportional to the degree of purification of PLA2 activity, (ii) the relative staining intensity of the 100-kDa protein precisely paralleled the elution profile of PLA2 activity during chromatography steps, (iii) the PLA2 activity recovered from a nondenaturing gel (greater than 60% of the total activity applied) coincided exactly with the major high molecular weight protein detected by silver staining, and (iv) monoclonal antibodies against the 100-kDa protein immunoprecipitated the PLA2. We conclude that the cytosolic PLA2 isolated from U937 cells represents a novel, high molecular weight PLA2 responding to physiological (intracellular) changes in Ca2+ concentration and therefore may play a critical role in cellular signal transduction processes and the biosynthesis of lipid mediators.     

Activation of myelin basic protein kinases during echinoderm oocyte maturation and egg fertilization

At least five activated protein kinases were detectable in soluble extracts from maturing as compared to immature sea star oocytes. These kinases could be distinguished on the basis of the time courses of their activation following exposure of the oocytes to 1-methyladenine, their substrate specificities, and their chromatographic properties on DEAE-Sephacel and Sephacryl S-200. A histone H1 kinase (HH1K) (Mr 110,000) underwent maximal activation near the time of 1-methyladenine-induced germinal vesicle breakdown (GVBD). When myelin basic protein (MBP) was used as a substrate, HH1K and two additional kinases (MBPK-I and MBPK-II) were detectable. MBPK-II (Mr 110,000) was fully activated at the time of GVBD, whereas peak activation of MBPK-I (Mr 45,000) occurred after this event. Two "ribosomal protein S6 kinases" (S6K-I and S6K-II) could be detected with a synthetic peptide (RRLSSLRA), which was patterned after a major phosphorylation site in S6. The two S6 kinases (Mr 110,000 for both) underwent activation post-GVBD. HH1K and S6K-I coeluted from DEAE-Sephacel at a conductivity of 5.5-6.0 mmho, whereas MBPK-I, MBPK-II, and S6K-II coeluted from this resin in a second peak at a conductivity = 10-11 mmho. The HH1K and MBPK-II activities both declined prior to the emission of the first polar body (i.e., meiotic cell division), but the MBPK-I, S6K-I, and S6K-II activities remained elevated during this time. The activities of these kinases were also examined during the early cell divisions in sea urchin embryos. Within 5 min after fertilization, the high level of MBPK-I activity in sea urchin eggs rapidly declined. However, along with the HH1K and MBPK-II activities, the MBPK-I activity was transiently increased prior to each cell division. No appreciable postfertilization changes in the S6K-I and S6K-II activities were apparent during the first three cycles of cell division.     

Identification and characterization of phospholipase A2 inhibitory proteins in human mononuclear cells

Calcium-dependent phospholipid binding and phospholipase A2 inhibitory proteins were isolated from human mononuclear cells. Lipocortins I and II were present whereas lipocortin IV (endonexin I) was not. The other proteins were purified to homogeneity and shown to have molecular masses of 35, 36, 32 and 73 kDa. The 36-kDa and 73-kDa proteins are related, the smaller appears to be part of the larger. The 73-kDa protein is related to the 67-kDa calelectrin and to lipocortin VI; the 32-kDa protein is different from endonexin I but related to chromobindin 7 and to lipocortin V. The 35-kDa protein has been identified by tryptic peptide sequencing as lipocortin III. All these proteins inhibit phospholipase A2 activity in vitro and the three smaller ones inhibit the [3H]arachidonic acid release from prelabelled monocytes induced by the calcium ionophore A23187 in a dose-dependent manner.     

Porcine pancreatic phospholipase A2 stimulates secretin release from secretin-producing cells

We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

Kinetics of the hydrolysis of micellar substrates catalyzed by snake venom phospholipases A2.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by a cobra (Naja naja atra) (Group I) and a Habu (Trimeresurus flavoviridis) (Group II) PLA2s, were studied and compared with the results reported for other Group I and II enzymes. The substrate bindings to Group I enzymes were independent of the Ca2+ binding, whereas the substrate bindings to Group II enzymes were facilitated more than 10 times by the Ca2+ binding to the enzymes. The result for Group II enzymes, but not Group I enzymes, seemed compatible with the hypothesis for interpreting the catalytic mechanism that an intermediate complex should be stabilized by the coordination of the bound Ca2+ with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. The pH dependence of the kinetic parameters for the hydrolysis of the mixed micellar diC16PC, catalyzed by the cobra (N. naja atra) (Group I) and Habu (T. flavoviridis) (Group II) PLA2s, was also studied. The pK values of the catalytic group, His 48, and Tyr 52 for N. naja atra PLA2, shifted from 7.25 to 7.70 and from 10.30 to 10.85, respectively, and the corresponding values for T. flavoviridis PLA2 shifted from 5.80 to 6.95 and from 10.10 to 10.76, respectively, on binding of the micellar substrates to the enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of three forms of phosphorylase and phosphorylase kinase from human skeletal muscle]

Three forms of phosphorylase (I, II and III), two of which (I and II) were active in the presence of AMP and one (III) was active without AMP, were isolated from human skeletal muscles. The pI values for phosphorylases b(I) and b(II) were found to be identical (5.8-5.9). During chromatofocusing a low molecular weight protein (M(r) = 20-21 kDa, pI 4.8) was separated from phosphorylase b(II). This process was accompanied by an increase of the enzyme specific activity followed by its decline. During reconstitution of the complex the activity of phosphorylase b(II) returned to the initial level. Upon phosphorylation the amount of 32P incorporated into phosphorylase b(II) was 2 times as low as compared with rabbit phosphorylase b and human phosphorylase b(I). It may be supposed that in the human phosphorylase b(II) molecule one of the two subunits undergoes phosphorylation in vivo. This form of the enzyme is characterized by a greater affinity for glycogen and a lower sensitivity to allosteric effectors (AMP, glucose-6-phosphate, caffeine) compared with phosphorylase b(I). Thus, among the three phosphorylase forms obtained in this study, form b(II) is the most unusual one, since it is partly phosphorylated by phosphorylase kinase to form a complex with a low molecular weight protein which stabilizes its activity. A partially purified preparation of phosphorylase kinase was isolated from human skeletal muscles. The enzyme activity necessitates Ca2+ (c0.5 = 0.63 microM). At pH 6.8 the enzyme is activated by calmodulin (c0.5 = 15 microM). The enzyme activity ratio at pH 6.8/8.2 is equal to 0.18.     

Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.     

Generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells

Conditions that regulate the generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in cultured rat cerebellar granule cells have been investigated. Under basal conditions, 4-5% of total CaM-kinase II activity, assayed in the presence of Ca2+/CaM, was the Ca2(+)-independent form active in the presence of EGTA. Depolarization with 56 mM K+ produced a transient increase to 9% Ca2+ independence within 15 s followed by a decline to 5-6% at 10 min. The divalent cation ionophore ionomycin elicited 10% Ca2+ independence, which remained elevated. Removal of Ca2+ from the Krebs-Ringer medium reduced basal Ca2+ independence to 1-2% and eliminated the elevation in response to K+ depolarization. Inclusion of 5 microM okadaic acid, a protein phosphatase inhibitor, in the incubation medium potentiated the levels of Ca2(+)-independent activity of CaM-kinase II. Additional studies in granule cell extracts indicated that there were both okadiac acid-sensitive and -insensitive protein phosphatases involved in the reversal of the Ca2+ independence of CaM-kinase II. Phosphopeptide mapping of the CNBr-cleaved 32P-labeled 58-60-kDa subunit of CaM-kinase II revealed that under basal conditions, the kinase contained phosphate in many sites. Conditions that promoted formation of the Ca2(+)-independent form of the kinase increased the 32P incorporation into multiple sites of the kinase. However, there was a good temporal correlation between 32P incorporation into CNBr peptide 1, which contains Thr-287, and generation of the Ca2(+)-independent kinase activity. These results indicate that formation of the Ca2(+)-independent species of CaM-kinase II is dynamically regulated in cerebellar granule cells by Ca2(+)-mobilizing agents and by protein phosphatase activity and is correlated with autophosphorylation of Thr-287.     

Calpactin-like proteins in human spermatozoa

Polyclonal antibodies directed against human calpactin I (p36) and calpactin II (p35) have been employed to investigate the distribution of calpactin-like proteins in human spermatozoa. Calpactins are a family of Ca2+-regulated cytoskeletal proteins that are major substrates of oncogene and growth factor receptor protein tyrosine kinases. The existence of a Triton-soluble 37-kDa protein antigenically related to calpactin II from somatic cells was revealed by Western blot analysis of human sperm extracts. The 37-kDa protein was not released from spermatozoa after experimental induction of the acrosome reaction by A23187 and Ca2+. Treatment of sperm homogenates with an EGTA-containing buffer partially solubilized the 37-kDa protein from the corpuscolate matter. Indirect immunofluorescence microscopy showed that anticalpactin II binds specifically to the sperm tail and to a band-like structure encircling the sperm head at the equatorial segment. In contrast, antibodies to calpactin I were found to bind to the tail midpiece, but failed to bind to Western blots of sperm proteins. This is the first immunological and biochemical report on the presence of calpactin proteins in a germ cell, the human spermatozoon.     

Expression and characterization of calmodulin-dependent protein kinase II from cloned cDNAs in Chinese hamster ovary cells

cDNAs containing the entire coding regions of the alpha and beta subunits of calmodulin-dependent protein kinase II (CaM kinase II) were isolated from a rat cerebrum cDNA library, ligated into an expression vector under the control of SV40 early promoter and introduced into Chinese hamster ovary (CHO) cells. To investigate the role of the alpha and beta subunits and their functional domains in CaM kinase II activity, the properties of the kinases expressed in the transfected cells were studied. CaM kinase II activity was detected in the transfected cells when the alpha and beta cDNAs were introduced into CHO cells simultaneously. RNA transfer blot and protein immunoblot analyses demonstrated the expression of the mRNAs and proteins of both alpha and beta subunits in the cloned cells. When alpha or beta cDNA was introduced into CHO cells separately, a significant level of the enzyme activity was also expressed, indicating that the alpha and beta subunits exhibited enzyme activity individually. The apparent Km values for ATP and MAP 2 were almost the same for the alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II. However, there was a slight difference in the affinity for calmodulin between the expressed proteins. The alpha and beta subunits expressed in the same cells polymerized to form alpha beta complex of a size similar to that of brain CaM kinase II. The alpha subunit also polymerized to form an oligomer, which showed almost the same S value as that of alpha beta complex and brain CaM kinase II. In contrast, the beta subunit did not polymerize. The alpha subunit, beta subunit, alpha beta complex, and brain CaM kinase II were autophosphorylated with [gamma-32P]ATP in the presence of Ca2+ and calmodulin, which resulted in the appearance of Ca2+-independent activity. The Ca2+-independent activity was 60-75% of the total activity as measured in the presence of Ca2+ plus calmodulin. To examine the functional relationship of peptide domains of the subunits of CaM kinase II, deleted cDNAs were introduced into CHO cells and the properties of the expressed proteins were studied. In cells transfected with alpha or beta cDNA from which the association domain was deleted, a significant level of kinase activity was expressed. However, the expressed proteins showed hardly any autophosphorylation and the appearance of Ca2+-independent enzyme activity was very low, indicating that the association domain was essential for the autophosphorylation and for the appearance of the Ca2+-independent activity.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue

Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.     

Structural and immunological properties of the phenolic glycolipids from Mycobacterium gastri and Mycobacterium kansasii.

Mycobacterial species-specific antigens belong to the three following classes: phenolic glycolipids (Phe Gl), acyltrehalose-containing lipooligosaccharides and polar glycopeptidolipids. These antigens have been chemically defined and alkali-labile epitopes were found to characterize the lipooligosaccharide antigen type. In the present study the major Mycobacterium kansasii phenolic glycolipid epitope namely Phe Gl K-I was delineated as the distal monoacetylated disaccharidic residue: 2,6-dideoxy-4-O-methyl-alpha-D-arabino-hexopyranosyl-(1----3)-2-O-methyl -4-O- acetyl-alpha-L-fucopyranose. This acetoxy group is required for K-I epitope recognition demonstrating that alkali-labile epitopes also occur in the phenolic glycolipid antigen class. Using immunoelectron microscopy, the Phe Gl K-I epitope was localized around the electron-transparent layer on the M. kansasii cell-wall surface. Furthermore, two new phenolic glycolipids namely Phe Gl K-III and Phe Gl K-IV were discovered in minute amounts. They were purified and characterized by their retention time in direct-phase column HPLC. These molecules are also M. kansasii antigens, whose epitopes differ from that of Phe Gl K-I. The complete family of phenolic glycolipids Phe Gl K-I, K-II, K-III and K-IV was found in both rough and smooth variants of both M. kansasii and Mycobacterium gastri species.     

Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors

Reversible calcium-dependent association with a particulate fraction from human placenta was used as the first step in the purification of substrates for the epidermal growth factor-stimulated protein kinase. A protein with apparent Mr of 35,000 was purified to homogeneity, and the sequence was determined for approximately one-fourth of the protein. These residues could be aligned exactly with the previously published sequence of lipocortin I derived from the cDNA from a human lymphoma. Two other proteins that appear to be formed by proteolytic removal of 12 or 26 of the amino acids from the NH2 terminus of the protein also were isolated. Placental lipocortin I was phosphorylated in Tyr-21 in an epidermal growth factor-dependent manner by the kinase activity in a particulate fraction from A431 cells; half-maximal phosphorylation occurred at 50 nM lipocortin I. Lipocortin I phosphorylated on Tyr-21 was approximately 10-fold more sensitive to tryptic cleavage at Lys-26 than was the native protein. Placental lipocortin I and its two truncated forms were potent inhibitors of pancreatic phospholipase A2 activity. Another 33-kDa protein that was not related immunologically to lipocortin I or lipocortin II (calpactin I) also was purified from the EGTA extract of placenta. The unidentified protein inhibited phospholipase A2 but was not a substrate for the epidermal growth factor-stimulated kinase. The mechanism by which these proteins inhibit phospholipase A2 activity was investigated. Attempts to detect direct interaction between these proteins and the enzyme were unsuccessful. However, both the unidentified protein, lipocortin I, and 32P-labeled lipocortin I bound in a Ca2+-dependent manner to the [3H]oleic acid-labeled Escherichia coli membranes used as substrate in the phospholipase A2 assay. Heparin, which is known to block lipocortin I inhibition of phospholipase A2, also blocked binding of lipocortin I to E. coli membranes. The results of these and other experiments raise the possibility that placental lipocortin I inhibits phospholipase A2 activity in this assay by coating the phospholipid and thereby blocking interaction of enzyme and substrate.