Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “insertion”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,567 records · Page 87Linked to original sources
PubMed1999

Intracoronary shunt insertion prevents myocardial stunning in a juvenile porcine MIDCAB model absent of coronary artery disease.

OBJECTIVE: The relevance of regional LV myocardial ischemia/reperfusion induced by temporary left anterior descending (LAD) coronary artery occlusion during minimally invasive direct coronary artery bypass (MIDCAB) grafting is controversial. The purpose of our study was (1) to determine the impact of conventional LAD occlusion during left internal thoracic artery (LITA)-LAD anastomosis on regional LV myocardial ischemia and function, and (2) to evaluate if intra-LAD shunt insertion during LITA-LAD anastomosis prevents potential regional LV ischemia and dysfunction in a pig model. METHODS: In 20 anesthetized, mechanically ventilated pigs we performed LITA-LAD anastomosis on the beating heart without cardiopulmonary bypass during either 15 min LAD occlusion (occlusion-group; n = 10) or 15 min intra-LAD shunt insertion to maintain blood supply to the myocardium beyond the anastomosis (shunt-group; n = 10). Besides standard hemodynamics we determined the global and regional LV wall motion score index (WMSI) using epimyocardial echocardiography. To quantitate structural myocardial alteration we determined the inducible heat-shock protein-70 (HSP-70) in LV anterior wall myocardial biopsies. Data were recorded at baseline, at 15 min of LAD occlusion or shunt insertion, respectively, and at 30 min of reperfusion. At the end of the experiments we determined myocardial adenine nucleotide (ATP, ADP, AMP) and glycogen content. RESULTS: In both groups WMSI was not significantly different at 15 min LAD occlusion or shunt insertion, respectively, as compared to baseline. However, at 30 min reperfusion both global and regional WMSI demonstrated significant LV dysfunction in the occlusion-group, whereas LV function in the shunt-group remained normal. This was associated with higher myocardial HSP-70 expression in the occlusion-group (P < 0.05). Myocardial adenine nucleotide and glycogen contents were significantly better preserved in the shunt-group. CONCLUSIONS: Our data show that in a porcine MIDCAB model 15 min LAD occlusion and 30 min reperfusion result in significant myocardial stunning. In contrast, maintenance of LAD perfusion using intracoronary shunt insertion minimizes ischemia/reperfusion injury and prevents regional LV dysfunction. Although our experiments were conducted in healthy pig hearts absent from coronary artery disease, similar results may--at least partially--be expected in humans, and thus, intracoronary shunts could be a useful tool for myocardial protection during 'off-pump revascularization'.

Adenine Nucleotides↗
PubMed2001

Air embolism during insertion of central venous catheters.

PURPOSE: To determine the clinical consequences of air embolism occurring during insertion of central venous catheters. MATERIALS AND METHODS: A computer search of our interventional radiology database revealed that 11,583 central venous catheters were inserted between January 1, 1995 and August 1, 2000: 7,178 were nontunneled and 4,404 were tunneled. Air embolism was reported to have occurred in 15 patients. Air embolization was recognized by audible air aspiration during catheter insertion or by fluoroscopic visualization of air within the right atrium or pulmonary artery. The radiology reports and medical records of these 15 patients were reviewed. RESULTS: All 15 patients had an air embolism occur during insertion of a tunneled central venous catheter. These included eight Ash catheters, five chest wall ports, one Tesio catheter, and one Pheres-Flow catheter. Four patients remained asymptomatic. Six patients had mild symptoms that quickly resolved with supplemental oxygen. Four had moderate symptoms that also resolved with supplemental oxygen. One patient died acutely as a result of the air embolism. CONCLUSION: Air embolism is a rare but potentially fatal complication of central venous catheter procedures. In our series, all occurred during insertion of a tunneled catheter through a peel-away sheath. The administration of supplemental oxygen was an effective treatment in the majority of patients.

Adult↗
PubMed1995

Extraumbilical insertion of the operative laparoscope in patients with extensive intraabdominal adhesions.

In 12 patients who had known or suspected intraabdominal adhesions, we evaluated the benefit of using Palmer's point for inserting the Veress needle and primary cannula during laparoscopic adhesiolysis to prevent direct intestinal injury. After inserting the laparoscope through this point, four women were found to have extreme intestinal adhesions around the umbilical area, and intestinal or severe omental injury might occur if the cannulas were inserted directly through the umbilical fossa. The other eight patients had omentum, partial to severe, adherent to the anterior abdominal wall. No complication occurred during insertion of the Veress needle and primary cannula through Palmer's point. Nine patients had successful laparoscopic adhesiolysis and were discharged within 2 days. In the other three women the procedure was converted to laparotomy because of extensive intestinal adhesions (2 patients) and small bowel injury during laparoscopic adhesiolysis (1). Palmer's point can be considered a safe and good alternative site for inserting the Veress needle and primary cannula to lyse dense intestinal and extensive omental adhesions. This technique should provide the surgeon with wider visual angel and surgical field, thus making adhesiolysis much easier to perform.

Abdomen↗
PubMed1999

Minilaparoscopy to reduce complications from cannula insertion in patients with previous pelvic or abdominal surgery.

STUDY OBJECTIVE: To evaluate the safety and efficacy of minilaparoscopy for visualization and adhesiolysis of intraabdominal adhesions for safe insertion of a primary umbilical cannula in patients who had previous pelvic or abdominal surgery. DESIGN: Prospective, observational study (Canadian Task Force classification II-1). SETTING: University-affiliated hospital. PATIENTS: Twenty women who had previous pelvic or abdominal surgery, excluding tubal ligation and simple appendectomy. INTERVENTION: Minilaparoscopy was carried for visualization and adhesiolysis at the entry site before insertion of an umbilical cannula. MEASUREMENTS AND RESULTS: Eleven patients had Pfannenstiel scars and nine had vertical midline scars from previous surgeries (4 total abdominal hysterectomies, 13 adnexal surgery, 7 cesarean sections, 3 ectopic pregnancies, 1 ruptured appendix, 1 unknown bowel surgery). Thirteen women (65%) had had one previous surgery, five (25%) had had two, and two (10%) had had three. Nine patients (45%) had significant adhesions of omentum or intestine that required adhesiolysis. Mean operating time for minilaparoscopy was 5 minutes for 11 women who had no or minimal adhesions that did not interfere with safe insertion of the umbilical cannula and effective use of a 10-mm telescope, and 25 minutes in 9 patients who required adhesiolysis before insertion of the umbilical cannula because of dense adhesions of omentum or intestine at the entry site. No complications with minilaparoscopy occurred. CONCLUSION: Minilaparoscopy can be performed safely and effectively to reduce serious vascular or visceral injury from insertion of primary cannula in patients who had previous pelvic and/or abdominal surgery. However, conclusions cannot be derived from this study due to the small number of subjects. Further study is necessary in a larger patient population to evaluate efficacy, safety, and advantages of minilaparoscopy over other techniques. (J Am Assoc Gynecol Laparosc 6(1):91-95, 1999)

Abdomen↗
PubMed1997

Morphometric analysis of the insertion of the upper head of the lateral pterygoid muscle.

OBJECTIVE: This article reports on the morphometric characteristics of the upper head of the lateral pterygoid muscle. STUDY DESIGN: Several morphologic characteristics were initially examined in 40 postmortem specimens. The length of the upper head of the lateral pterygoid muscle was determined in 34 specimens. Of the 26 specimens in which the upper head of the lateral pterygoid muscle was found to be inserted into the meniscus and condyle, 22 specimens were examined microscopically to morphometrically quantify the proportion of muscle fibers to their respective insertions. RESULTS: The upper head of the lateral pterygoid muscle was approximately 33.3 mm in length and was significantly longer in males. There were no age-related differences. Approximately 29.5% of the fibers of this muscle were inserted into the meniscus of the temporomandibular joint. CONCLUSION: This study demonstrated that the insertion of the upper head of the lateral pterygoid muscle to the meniscus ranged from a fleshy mass to a few fibers. The number of fibers entering both the meniscus and the condyle decreased correspondingly as they approached the joint and could be interpreted as this muscle having a tendon of insertion into the meniscus.

Adult↗
PubMed1998

T. brucei RNA editing: adenosine nucleotides inversely affect U-deletion and U-insertion reactions at mRNA cleavage.

In the currently envisioned mechanism of trypanosome mitochondrial RNA editing, U-insertion and U-deletion cycles begin with a common kind of gRNA-directed cleavage. However, natural, altered, and mutationally interconverted editing sites reveal that U-deletional cleavage is inefficient without and activated by ATP and ADP, while U-insertional cleavage shows completely reverse nucleotide effects. The adenosine nucleotides' effects appear to be allosteric and determined solely by sequences immediately adjacent to the anchor duplex. Both U-deletional and U-insertional cleavages are reasonably active at physiological mitochondrial ATP concentration. Notably, ATP and ADP markedly stimulate complete U-deletion and inhibit U-insertion reactions, reflecting their effects on cleavage. These plus previous results suggest that U deletion and U insertion are remarkably distinct.

Adenosine↗
PubMed1989

Insertion-deletion variation at the yellow-achaete-scute region in two natural populations of Drosophila melanogaster.

We have surveyed the region of the X chromosome of Drosophila melanogaster which encodes the yellow, achaete and scute genes for restriction map variation. Two natural populations, one from North Carolina, U.S.A. and the other from southern Spain were screened for variation at about 70 restriction sites and for variation due to DNA insertion or deletion events in 120 kilobases of DNA. Mean heterozygosity per nucleotide was estimated to be 0.0024 and 15 large insertions were found in the 49 chromosomes screened. Extensive disequilibrium between polymorphic sites were found across much of the region in the North Carolina population. The frequency of large insertions, which usually correspond to transposable genetic elements, is significantly lower than has been observed in autosomal regions of the genome. This is predicted for X-linked loci by certain models of transposable element evolution, where copy number is restricted by virtue of the recessive deleterious effects of the insertions. Our results appear to support such models. The deficiency of insertions may in this case be enhanced by hitch-hiking effects arising from the high level of disequilibrium.

Animals↗
PubMed1991

Transposable elements in natural populations with a mixture of selected and neutral insertion sites.

This paper examines models of the population dynamics of transposable elements when chromosomal sites vary with respect to the effect on fitness of mutations caused by element insertions. Element abundance is assumed to be stabilised solely by the joint results of transposition, excision, and selection against insertional mutations. When there are only two classes of site, selected and neutral, it is hard to find parameter values for which numbers of elements are maintained that match the findings from surveys of Drosophila populations, as elements tend to accumulate at high frequencies at the neutral sites. It is similarly hard to produce realistic equilibria with three classes of site (strongly selected, weakly selected, and neutral), when elements can transpose out of the neutral sites. If transposition from neutral sites is impossible, as might be the case for elements inserted into centric heterochromatin, then realistic equilibria can be generated if there is very weak selection against elements inserted into the majority of non-neutral sites. This model predicts a modest over-representation of elements at the neutral sites. It also predicts that elements should be under-represented on the X chromosome compared with the autosomes, but this is not generally found to be the case. It is concluded that selection against insertional mutations is unlikely to be the major factor involved in the containment of element abundance.

Animals↗
PubMed1998

Grommet insertion in children: a survey of parental perceptions.

Grommet insertion is a widely accepted method of treatment of glue ear in children. There have been questions raised over the last few years about the indications for grommets and whether assessing the hearing alone is an efficient outcome measure. Parental pressure accounts for one of the factors that is taken into consideration when the decision to insert grommets for glue ear is made. In this paper, a prospective questionnaire is used to investigate the parental perceptions of the effectiveness of grommet insertion in children, focusing on alternative outcome measures such as general health, language, and social skills. The results of this survey suggest that grommet insertion causes improvement in many factors other than hearing and this seems to account for the parental pressure for siblings to have grommet insertion.

Attitude↗
PubMed2006

Should we aspirate middle-ear effusions prior to insertion of ventilation tubes?

BACKGROUND: Routine aspiration of middle-ear effusions prior to ventilation tube (grommet) insertion is practised by many surgeons. It has been suggested that removing the fluid from the middle ear improves immediate post-operative hearing levels and reduces the chance of the ventilation tube becoming obstructed. The potential adverse effects of applying suction to the middle ear include acoustic trauma and an increased risk of tympanosclerosis and otorrhoea. We undertook a review of the literature in order to determine the benefits or side effects associated with middle-ear aspiration prior to ventilation tube insertion. OBJECTIVES: To compare clinical outcomes associated with aspirating versus not aspirating the middle ear prior to ventilation tube insertion. METHODS: The Cochrane ENT group trials register, DARE, the Cochrane central register of controlled trials (CENTRAL), MEDLINE (1960-2005) and EMBASE (1960-2005) were searched using relevant terms. Reference lists of selected studies were scanned for additional research material. RESULTS: Seven studies were identified, of which three fitted the inclusion criteria of our review. Current evidence suggests that aspiration of middle-ear effusions prior to insertion of ventilation tubes is not associated with any improvement in clinical outcome, in terms of post-operative hearing levels, otorrhoea or rates of blockage of ventilation tubes. Significantly increased rates of tympanosclerosis were observed in one study and the development of acoustic trauma was observed; however, no significant association was confirmed. Although more research is needed, there is no evidence that aspiration of middle-ear effusion prior to grommet insertion confers any advantage.

Hearing Loss, Noise-Induced↗
PubMed2002

Conditions for insertion of the laryngeal mask airway: comparisons between sevoflurane and propofol using fentanyl as a co-induction agent. A pilot study.

BACKGROUND AND OBJECTIVE: To compare the conditions for insertion of the laryngeal mask airway using sevoflurane or propofol plus fentanyl. We evaluated the haemodynamic changes and cost of induction of anaesthesia in both groups. METHODS: Sixty patients were equally and randomly divided into two groups. Both groups received fentanyl 1 microg kg(-1). Patients in the sevoflurane group were induced with 8% sevoflurane and those in the propofol group with propofol 2.5 mg kg(-1). Conditions for insertion were graded on a three-point scale using six variables. Overall, conditions were assessed as excellent, satisfactory or poor based on the total score in each group. Systolic and diastolic arterial pressure and heart rate were recorded for 6 min after mask insertion. The financial cost of induction in both groups was calculated. RESULTS: The mean (+/- SD) time taken from induction to successful laryngeal mask insertion was significantly shorter with propofol (68.70 +/- 22.60 s) compared with sevoflurane (149.83 +/- 55.25 s). Excellent or satisfactory conditions were observed in 30 (100%) patients in the propofol group and in 29 (96.66%) in the sevoflurane group. Systolic and diastolic arterial pressures were significantly lower in the propofol group. The cost of sevoflurane used was 3.95 euros +/- 1.48 (Rs 216.23 +/- 64.66) (P < 0.05) compared with that of propofol, which was 3.23 euros +/- 0.65 (Rs 141.00 +/- 28.20). CONCLUSIONS: Although there was a faster induction with propofol-fentanyl, conditions for insertion of the laryngeal mask airway were similar in both groups. Haemodynamic stability was better with sevoflurane-fentanyl. The propofol-fentanyl combination was more cost-effective.

Adult↗
PubMed2005

Lumbar epidural catheter insertion: the midline vs. the paramedian approach.

BACKGROUND AND OBJECTIVE: The purpose of this study was to compare the characteristics of epidural catheter insertion via the midline or the paramedian approach with regard to ease of catheter insertion, incidence of paraesthesias and efficacy of epidural block. In addition to the type of approach, the prognostic value of Patients characteristics variables with regard to the incidence of paraesthesias was assessed. METHODS: Thirty patients scheduled for surgery under epidural anaesthesia were randomly assigned to one of two groups of 15 patients each. Epidural anaesthesia was performed via a midline or paramedian approach using loss of resistance to saline. Variables measured were: time needed to identify the epidural space, time needed for and ease of epidural catheter insertion and the incidence of paraesthesias. After completion of these observations, epidural anaesthesia was established with 150 mg ropivacaine 1%. Efficacy of the epidural block was assessed by the need for intraoperative analgesics and by the patient on a three-point scale (good/fair/poor). RESULTS: Quality of sensory blockade was adequate in both groups. Catheter insertion was significantly faster using the paramedian approach. The difference between the two approaches with regard to the incidence of paraesthesias was not significant, however, there was a trend towards more paraesthesias in the midline group. In the multivariate analysis, type of approach was an independent significant predictor of paraesthesias and we found a trend towards a higher incidence of paraesthesias in female patients. CONCLUSIONS: Catheter insertion was faster in the paramedian group and we found a trend towards a higher incidence of paraesthesias with the midline approach.

Anesthesia, Epidural↗
PubMed1995

Membrane topology of P-glycoprotein as determined by epitope insertion: transmembrane organization of the N-terminal domain of mdr3.

P-Glycoproteins (P-gps) are membrane glycoproteins encoded by the mdr gene family, and their overexpression is associated with multidrug resistance (MDR). Sequence analyses of mdr cDNAs predict a protein formed by two symmetrical halves, each composed of six transmembrane (TM) segments and one ATP-binding domain. To determine the topology of the N-terminal half of P-gp, a small antigenic peptide epitope (YPYDVPDYAIEGR) containing part of the hemagglutinin (HA) of influenza virus was inserted at six different positions of the Mdr3 protein (101, 161, 206, 244, 320, and 376). Functional integrity of the modified proteins was tested by measuring their capacity to confer MDR in Chinese hamster ovary cells. Intracellular and extracellular localization of the tag in the full-length protein was determined in intact or permeabilized cells by immunofluorescence using a mouse monoclonal antibody (12CA5) specific for the HA epitope. While insertions at positions 101, 161, 320, and 376 did not alter P-gp function, insertions at positions 206 and 244 abrogated the capacity of P-gp to confer drug resistance. The epitope tags inserted at positions 161 and 376 were found to be located intracellularly, whereas the tags at positions 101 and 320 were located on the extracellular side of the membrane. These results indicate that the intervening segments separating predicted TM1-TM2 and TM5-TM6 correspond to extracellular regions, while the segments linking TM2-TM3 and the one located downstream of TM6 correspond to intracellular regions. These results are consistent with a six TM domain model for the N-terminal half of P-gp with an extracellular glycosylated region (TM1-TM2) and an intracellular ATP-binding site (downstream TM6). Epitope insertion in segments linking TM3-TM4 and TM4-TM5 caused a loss of P-gp function, suggesting that the integrity of these sequences is essential either for drug transport or for proper maturation and accurate targeting of P-gp to the plasma membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins.

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning beta-sheet. A PFO oligomeric complex was formed on liposomes at both 4 degrees C and 37 degrees C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its beta-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane beta-sheet.

Bacterial Toxins↗
PubMed1993

Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases.

Identical or highly similar antigenic determinants, not present in the intact inhibitor, were induced in antithrombin on cleavage of the reactive bond, on formation of a complex between antithrombin and a synthetic reactive-loop tetradecapeptide, and on partial denaturation of antithrombin at low concentrations of guanidinium chloride. Previous studies indicate that the common structural feature of these three modified forms of antithrombin is that the region of the reactive-bond loop on the amino-terminal side of the reactive bond, or the corresponding synthetic peptide, is inserted as a middle strand in the main beta-sheet of the inhibitor, the A sheet. The new epitopes in the three modified antithrombin forms therefore most likely are exposed as a result of this insertion. Identical or highly similar epitopes were exposed also in complexes between antithrombin and thrombin or factor Xa, strongly suggesting that a substantial segment of the reactive-bond loop is inserted into the A sheet also in these complexes. In contrast, the new epitopes were not exposed in antithrombin on binding of heparin, implying that the conformational change induced by heparin does not involve such loop insertion. These results provide the first experimental verification of recent hypotheses that insertion of the reactive-bond loop of serpins into the A beta-sheet is involved in the binding of target proteinases.

Amino Acid Sequence↗
PubMed2000

Activated protein C-protein C inhibitor complex formation: characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop.

Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.

Animals↗
PubMed2000

Role of the beta-strand insert in the central domain of the fibrinogen gamma-module.

The crystal structure of the fibrinogen gamma-module (residues gamma143-411) [Yee, V. C., et al. (1997) Structure 5, 125-138] revealed an unusual feature. Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible. To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli. It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region. A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized. This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286. They all were folded, but those devoid of the beta-strand insert were destabilized. The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter. On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.

Calorimetry, Differential Scanning↗
PubMed1988

Properties of membrane-inserted protein kinase C.

Protein kinase C (PKC) interacted with phospholipid vesicles in a calcium-dependent manner and produced two forms of membrane-associated PKC: a reversibly bound form and a membrane-inserted form. The two forms of PKC were isolated and compared with respect to enzyme stability, cofactor requirements, and phorbol ester binding ability. Membrane-inserted PKC was stable for several weeks in the presence of calcium chelators and could be rechromatographed on gel filtration columns in the presence of EGTA without dissociation of the enzyme from the membrane. The activity of membrane-inserted PKC was not significantly influenced by Ca2+, phospholipids, and/or PDBu. Partial dissociation of this PKC from phospholipid was achieved with Triton X-100, followed by dialysis to remove the detergent. The resulting free PKC appeared indistinguishable from original free PKC with respect to its cofactor requirements for activation (Ca2+, phospholipid, and phorbol esters), molecular weight, and phorbol 12,13-dibutyrate (PDBu) binding. The binding of PDBu to free and membrane-inserted PKC was measured under equilibrium conditions using gel filtration techniques. At 2.0 nM PDBu, free PKC bound PDBu with nearly 1:1 stoichiometry in the presence of Ca2+ and phospholipid. No PDBu binding to the free enzyme was observed in the absence of Ca2+. In contrast, membrane-inserted PKC bound PDBu in the presence or the absence of Ca2+; calcium did enhance the affinity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗