Joan I. Smallwood

Analysis of the PKC-γ-Related Immunocrossreactive Region of a Novel Leukocyte Protein γ-rp

The newly discovered gamma-PKC-related-protein of human leukocytes (γ-rp) crossreacts with a polyclonal antibody preparation originally designed to be specific for PKC-γ (γMb-Ab). As this antibody is currently the only suitable probe for γ-rp, we sought to characterize the binding of the two proteins. We determined that the γMg-Ab does not recognize the native form of γ-rp. However, with denaturing immunoblots of γ-rp, we found that 1) the crossreactive γ-rp epitope differs somewhat from that of classic rat brain PKC-γ, but probably only to the degree of the rat/human PKC species difference; 2) the previously reported doublet bands of γ-rp represent a single protein with cell-stimulus inducible modifications; 3) antibodies present in the γMg-Ab pool bind to two separate sites within the γ-rp epitope; 4) access to one binding site is conformationally restricted, even after protein denaturation; 5) agonist-induced modification of γ-rp does not significantly affect the total amount of γMg-Ab that it can bind, but 6) does significantly affect the rate of antibody binding to one site. This investigation defines the appropriate experimental use of our antibody, and the significance of these findings for the future study and cloning of γ-rp is discussed.

Protein kinase C isoforms in human neutrophil cytoplasts.

Granule‐poor human neutrophil cytoplasts, prepared without heat or cytochalasin B treatment so as to preserve both motile function and activatable respiratory burst oxidase, were investigated for their content of several isoforms of protein kinase C (PKC). Immunoblotting with isoform‐specific rabbit antibodies (Abs) to PKC revealed that both the α‐specific and the β(I and II)‐specific Abs recognized a protein band of 78 kd comigrating with PKC from rat brain cytosol. The γ‐specific antiserum did not detect any protein of this molecular mass. The cytoplast β‐PKC band was more readily detected than the cytoplast α‐PKC band. Antibodies to βI‐ or β‐II‐specific PKC sequences showed the βII subtype to be the predominant form of β‐PKC, although some βI was also found. The identity of the 78‐kd cytoplast bands as PKC was established by the fact that phorbol ester treatment of intact cytoplasts induced translocation of the bands from cytosol to membrane fractions. However, whereas PKC specific activity was similar in cytoplast lysates and brain cytosol, immunoreactivity of cytoplast α‐ and β‐PKC bands was considerably less than that of rat brain. Hydroxylapatite chromatography of partially purified cytoplast PKC revealed two major peaks of PKC activity precisely coeluting with brain a‐ and β‐PKC and displaying comparable enzymatic activities despite the relatively weak immunoreactivity of cytoplast α‐ and β‐PKC. To our knowledge, this is the first demonstration that human neutrophil–derived cytoplasts contain α, βI, and βII forms of PKC and that each isoform translocates from cytosol to membrane upon exposure to phorbol ester at concentrations that induce superoxide production. In addition, our evidence raises the possibility that cytoplasts may also possess other isoforms of PKC that we are unable to detect with our α, β, and γ antibodies. Finally, the granule‐poor cytoplasts seem a particularly useful preparation in which to examine the role of individual PKC isoforms in neutrophil activation.

Vasopressin-induced activation of protein kinase C in renal epithelial cells.

Recent studies indicate that the actions of arginine vasopressin (AVP) and other agonists that stimulate electrogenic sodium transport in renal epithelial A6 cells are linked to a Ca(2+)-mobilizing signal transduction mechanism that involves generation of inositol trisphosphate. Since diacylglycerol is the other product in this pathway, studies were performed to determine the possible role of PKC in the stimulation of sodium transport. AVP induced a biphasic increase in diacylglycerol generation, characterized by an initial rapid rise and then a sustained elevation, and PKC activation, reflected by phosphorylation of a specific 80 kDa myristoylated alanine-rich PKC substrate (MARCKS). To determine the PKC isoform(s) involved in this process, immunoblot analysis was performed using antisera that recognize both classical PKC isoforms, XPKC-I and XPCK-II, cloned from Xenopus oocytes. The transcripts of both isoforms were expressed in the A6 cell. Since protein recognized by antisera was translocated from cytosol to the particulate fraction after exposure to AVP, one or both isoforms were activated in the A6 cell. Further studies showed that cyclohexyladenosine and insulin, additional agonists of sodium transport in A6 cells, also stimulated phosphorylation of MARCKS. These results argue that Ca(2+)-dependent PKC is involved in the action of AVP, and that of other agonists, which stimulate sodium transport.

Identification of a Novel Hepatic Calcium-Binding Protein

Calcium binding activity in the 100,000 X g supernatant of bovine liver has been isolated by a procedure involving DEAE cellulose and Sephadex G-100 chromatography. In addition to calmodulin, two new high affinity calcium binding proteins have been identified. On gel filtration chromatography these proteins migrate with apparent molecular weights of 83,700 and 51,400; whereas by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the two proteins migrate identically with Mr 63,000. In the presence of millimolar Mg2+, both proteins bind up to one mol Ca2+/mol protein. Half-maximal binding occurs at approximately 0.1 microM Ca2+. Amino acid compositional analysis reveals that both proteins are acidic, and contain about 40% glx and asx. Peptide mapping procedures suggest that these proteins may be highly homologous or multiple forms of a single protein. The results show the existence of calcium binding protein(s) other than calmodulin in hepatic cytosol.

Chemotactic activity of human blood leukocytes in plasma treated with EDTA: chemoattraction of neutrophils about monocytes is mediated by the generation of NAP-2

In slide preparations of human blood leukocytes in autologous plasma containing EDTA, many adherent monocytes are initially chemotactic for neutrophils (PMN). We have identified the chemotactic factor that they generate as neutrophil‐activating peptide‐2 (NAP‐2), as evidenced by distraction of the gradient by authentic human NAP‐2, the importance of platelets in the media, which elaborate the precursor of NAP‐2, and suppression of the chemotactic response by serine protease inhibitors, which would block the monocyte‐derived serine esterase that creates NAP‐2 from its immediate precursor. Consistent with this conclusion is inhibition of the chemotactic response to monocytes by agents that block CXCR2, the receptor that NAP‐2 uses. Later, when the monocyte moves from the center of chemoattraction, the activated PMN themselves, whose own chemotactic properties are enhanced in EDTA/plasma, appear to take over generation of the gradient, resulting in a prolonged ingress of PMN from outside the field (“second wave”). Chemoattraction by monocytes seems to be simply one way of stimulating the PMN, which, once activated, fail in EDTA/plasma to efficiently shut off their own chemoattraction for other PMN. We suggest that these exaggerated chemotactic effects are due to the loss of normal modulation by a regulatory factor(s) designed to keep the chemotactic response from getting out of hand—i.e., a tonic inhibitor of chemotaxis in plasma.

Identification of novel calcium binding proteins of heart and brain 100,000 X G supernatant

The chelex competitive calcium binding assay has been used to assay the calcium binding activity of the 100,000 X g supernatant of bovine heart and brain. Chromatography of brain 100,000 X g supernatant on diethylamino-ethyl (DEAE) cellulose reveals the presence of two peaks of calcium binding activity, peak I eluting at about 0.05 M NaCl and peak II at about 0.18 M NaCl. Chromatography of peak I on Sephadex G-150 resolves a major and a minor peak of calcium binding activity, at Mr 40,000 and Mr 150,000. Chromatography of peak II (0.18 M NaCl) on Sepharose 6B produces two peaks of calcium binding activity, a broad peak of calcium binding activity composed of two molecular weight species of Mr 230,000 and Mr 420,000, and a sharp peak of calcium binding activity with Mr 75,000. Chromatography of the 100,000 X g supernatant of bovine heart on DEAE Cellulose reveals two peaks of calcium binding activity. Chromatography of the lower ionic strength peak on Sephadex G-150 resolved major and minor peaks of calcium binding activity at Mr 65,000 and 150,000, respectively. The results of this study suggest the presence of several calcium binding proteins, other than calmodulin, in these tissues.

Misoprostol Stimulates cAMP Generation in Human Leukocytes: Synergy with Colchicine Suggests a New Potential for Established Drugs.

In isolated leukocytes, elevation of cAMP can inhibit various proinflammatory and immune functions. The prostaglandins E (PGEs) are known to stimulate leukocyte cAMP production, and for years they have been viewed as potential immunosuppressive and/or anti-inflammatory agents. However, their clinical use is severely limited by extreme metabolic instability and by poor oral absorption, which necessitates administration by infusion or injection. Misoprostol is a synthetic analog of PGE1 that is relatively stable and orally absorbable. We examined the effects of misoprostol on cAMP production in leukocytes, in view of the possibility that it mimics PGE1 and, thus, might represent a clinically useful immunosuppressive or anti-inflammatory drug. Our results indicate the following: (1) Misoprostol increases leukocyte cAMP production in a dose-dependent manner (∼ 20 nM to > 100 μM) and acts by stimulating adenylate cyclase. (2) Its potency and maximal effect are somewhat less than those of PGE1 (3) cAMP generation in response to either misoprostol or PGE1 is transient (in the presence of isobutylmethylxanthine to inhibit endogenous phosphodiesterases). (4) Misoprostols stimulation of adenylate cyclase is synergistically increased by pretreatment of cells with colchicine, a microtubule-disrupting agent that is currently used for prophylaxis and treatment of gout. (5) Colchicine acts by increasing the initial rate of cAMP production and not by prolonging the response to misoprostol. (6) A clinically relevant dose of colchicine (0.25 μM) is effective given sufficient pretreatment time. (7) Whereas a clinically relevant dose of misoprostol (3 nM) is ineffective alone, preexposure of cells to colchicine enables such a dose to stimulate cAMP generation significantly. The combination of misoprostol with colchicine might eventually prove useful in the therapy of immune or inflammatory disease.

The role of band III in calcium transport across the human erythrocyte membrane

cellular concentration (10-j M) [l-3]. Two models have been presented to explain the mechanism of Ca*+ translocation in erythrocyte plasma membranes. The first model [4,5] was developed from studies of Ca*+ transport into inside-out membrane vesicles (IOV), from which it was observed that the active transport of Ca*+ into IOV was stimulated by anions such as phosphate, chloride, acetate and sulfate, and that the stimulation of calcium transport by these anions was due to an active, calcium-dependent uptake of the anions into IOV. Since both Ca*+ and anion transport were blocked by agents that inhibit the membrane anion transport protein, band III, it was proposed that Ca*+ transport was electrogenic; i.e., the transport of Ca*+ into IOV was associated with the development of a positive membrane potential. This, in turn, drove the transport of anions into the IOV through band III. In later work, direct evidence was obtained for an increase in membrane potential as a consequence of the ATP-dependent transport of calcium [ 121. The second model of Recently Niggli et al. [7] presented evidence that band III inhibitors block the Ca*+ -ATPase directly. They call into question our previous results [4] and suggest that band III does not play a role in Ca* + transport. In order to define more precisely the role of anion transport in Ca2+ translocation, further investigation of the effects of band III inhibitors on calcium transport and Ca* + -ATPase activity were conducted.

An apparently novel protein of human leukocytes, reactive with an antibody to protein kinase C-gamma, is rapidly modified upon cell activation: initial characterization in neutrophils and their cytoplasts.

On immunoblots of human neutrophil cytoplasts (U-CYT), a previously undescribed 97kDa protein was revealed by intense and selective reaction with an antibody that was initially raised to recognize PKC-γ. Denoted “γ-rp” for gamma-related protein, this acidic cytosolic protein somewhat resembled the classic forms of PKC in several biochemical respects. Appearing as a doublet on low-percentage SDS-PAGE gels, both its mobility and staining pattern were rapidly altered by treatment of U-CYT with either phorbol ester or chemotactic peptide. Whole neutrophil γ-rp was detectable only after TCA precipitation of intact cells. It was also detectable in human platelets, lymphocytes, and neutrophil-like differentiated HL60 cells, but not in fibroblasts, erythrocytes, monocytes, or monocyte-like differentiated HL60 cells. Our data suggest that γ-rp merits further study as a potential participant in cellular activation, and as a possible structural or functional relative of PKC.