Species- and tissue-dependent diversity of G-protein beta subunit phosphorylation: evidence for a cofactor.

We previously reported that, in the membranes of HL-60 cells during activation of G-proteins, a phosphate transfer reaction occurs which involves transient G-protein beta subunit (G beta) phosphorylation [Wieland, Nürnberg, Ulibarri, Kaldenberg-Stasch, Schultz and Jakobs (1993) J. Biol. Chem. 268, 18111-18118]. Here, the generality of this phenomenon is evaluated by studying membranes of various tissues obtained from different mammalian species. All membranes tested expressed at least G beta 1 and G beta 2 subunits. Cell membranes from bovine and porcine brain and liver, rat brain and human blood cells exhibited predominantly G beta 1 or both subtypes at roughly equal concentrations. In contrast, significantly more G beta 2 immunoreactivity was detected in membranes from human placenta. Bovine and porcine liver membranes exhibited weak, G beta-specific immunoreactive signals. Conversely, these membranes showed the highest levels of G beta phosphorylation after incubation with [gamma-32P]GTP or 35S-labelled guanosine 5'-[gamma-thio]triphosphate. Interestingly, G beta-specific phosphorylation of membranes from human erythrocytes and platelets was very weak. G beta phosphorylation was confirmed by immunoprecipitation with G beta-specific antibodies, and the target amino acid was identified as histidine. On SDS/PAGE, phosphorylated or thiophosphorylated G beta-proteins differed in their apparent molecular size from unmodified G beta-proteins. Moreover, phosphorylated G beta-proteins differed in a species-dependent fashion in their electrophoretic mobility. Solubilization of membrane proteins with detergent did not abolish G beta phosphorylation. In contrast, reconstituted purified Gi/Go proteins showed no G beta phosphorylation. From these experiments we conclude that: (i) G beta phosphorylation represents a general phenomenon occurring in the cells of various species to different degrees, (ii) phosphorylated G beta-proteins exhibit species-dependent diverse electrophoretic mobilities, and (iii) G beta phosphorylation requires a membrane-associated cofactor(s) which is lost during routine G-protein purification.

Receptor-induced translocation of activated guanine-nucleotide-binding protein alpha i subunits to the cytoskeleton in myeloid differentiated human leukemia (HL-60) cells.

The regulation of the cytoskeletal localization of guanine-nucleotide-binding protein alpha i subunits by formyl peptide receptors was studied in myeloid differentiated human leukemia (HL-60) cells. Stimulation of formyl peptide receptors with N-formyl-Met-Leu-Phe (fMet-Leu-Phe) transiently increased the amount of alpha i subunits in the Triton X-100-insoluble cytoskeleton. Similar to the biphasic regulation of the actin content, fMet-Leu-Phe ( > or = 10 nM) rapidly increased the cytoskeletal alpha i content (about threefold at 30 s), which was followed by a rapid reversal to control levels. The formyl peptide receptor increased the cytoskeletal content of both alpha i subtypes, alpha i2 and alpha i3- present in HL-60 cells. In cells permeabilized with Staphylococcus aureus alpha-toxin, fMet-Leu-Phe increased binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to cytoskeletal proteins in a pertussis-toxin-sensitive manner, which was completely abolished by the F-actin-disrupting agent, cytochalasin B. Using the photoreactive GTP analogue, m-acetylanilido-GTP, the formyl peptide receptor-regulated GTP binding sites at the cytoskeleton were identified as 40-kDa proteins, the molecular size of alpha i subunits. Cytoskeleton prepared from stimulated cells did not exhibit increased GTP[S] binding, which suggests that activated alpha i subunits are translocated to the cytoskeleton. Finally, in alpha-toxin-permeabilized HL-60 cells, fMet-Leu-Phe and GTP[S] cooperatively stimulated actin polymerization. In conclusion, evidence is provided that chemoattractant receptors cause translocation of activated alpha i subunits to the cytoskeleton coincidentally with F-actin formation. The data therefore argue for a potential role of translocated alpha i subunits in the process of receptor-induced actin polymerization.

Characterization of the epitope recognized by a monoclonal antibody directed against the largest subunit of Drosophila RNA polymerase II.

The epitope recognized by monoclonal antibody MAb215 generated previously against Drosophila melanogaster RNA polymerase II was mapped to amino acid residues 806-820 of the largest, 215 kDa, subunit located in a region conserved within the largest subunits of pro- and eukaryotic RNA polymerases. The affinities of MAb215 and of a recombinant single-chain Fv fragment (scFv215) were determined for binding to the enzyme as well as the fusion protein and synthetic peptides used for epitope mapping. In addition, amino acid residues of the epitope important for binding to MAb215 were identified using peptides carrying single amino acid substitutions. The epitope is not involved in the polymerization reaction or the DNA unwinding process since no inhibitory effects of the monoclonal antibody were observed in nonspecific in vitro transcription using denatured calf thymus DNA or double stranded oligo dC-tailed T7 DNA as template. In contrast, MAb215 inhibits accurate in vitro transcription from the Krüppel gene promoter and from the adenovirus-2 major late promoter. Preincubation of template DNA with the nuclear extract had no effects on inhibition supporting the notion that the epitope does not participate directly in the formation of preinitiation complexes. The same inhibitory effects were observed using scFv215. The results provide further evidence that recombinant antibody fragments produced in Escherichia coli possess the same specificity and similar affinity as their parental antibodies and demonstrate that scFv fragments are useful tools for analysis of transcriptional processes.

RPII15 codes for the M(r) 15,000 subunit 9 of Drosophila melanogaster RNA polymerase II.

The RPII15 gene product of Drosophila melanogaster, which has recently been identified by sequence comparison, possesses a high similarity to subunit 9 of yeast RNA polymerase II. Using the polymerase chain reaction the coding region of RPII15 was isolated from genomic DNA of adult flies. Sequence analysis shows four amino acid substitutions in comparison to the previously reported sequence. Antisera were generated against bacterially expressed RPII15 and were used for immunoblotting experiments with RNA polymerase II of Drosophila melanogaster. This analysis identified the M(r) 15,000 subunit 9 as gene product of RPII15.

Predictors of clinical course, coronary anatomy and left ventricular function after recovery from acute myocardial infarction.

Patients who survive an acute myocardiac infarction (AMI) have significant coronary disease and are at risk for angina pectoris, recurrent myocardiac infarction and sudden death. This study provides data gathered prospectively for 106 patients surviving myocardial infarction who had coronary arteriography, left ventriculography and 24-hour electrocadiographic recordings before hospital discharge and were followed 30 months. Univariate analysis showed that low ejection fraction, proximal left anterior descending coronary disease and significant disease in all three coronary arteries were associated with a high risk of sudden cardial death. The ECG location or type of infarction was not helpful in predicting mortality, reinfarction or continuing angina. Multivariate analysis of 30 clinical and laboratory variables identified previous myocardial infarction and an ejection fraction less than 40% as the best predictors of mortality; all 13 patients who died were identified by these two variables. Three-vessel coronary artery disease, proximal left coronary disease and complicated late hospital-phase ventricular arrhythmias did not provide additional information about mortality once the information provided by the first two variables was considered. Multivariate analysis identified hypertension, three-vessel coronary disease, postinfarction angina pectoris and previous AMI as significant predictors of recurrent AMI during the 30 month follow-up.

Coronary angiography and left ventriculography in survivors of transmural and nontransmural myocardial infarction.

Recent studies have suggested a similar prognosis for patients with transmural myocardial infarction and nontransmural myocardial infarction despite a smaller infarct size in the latter patients estimated by creatine phosphokinase (CPK). Thirty-one patients with transmural myocardial infarction and 17 patients with nontransmural myocardial infarction as defined by electrocardiographic criteria underwent coronary angiography and left ventriculography from 10 to 24 days after they had an acute myocardial infarction. Forty-three of these 48 patients were asymptomatic following their myocardial infarction. When compared to patients with nontransmural myocardial infarction, those with transmural myocardial infarction had greater peak CPK levels, 1,090 +/- 210 versus 290 +/- 60 IU (p less than 0.01). There was no difference in prevalence of single, double or triple vessel coronary artery disease, mean number of coronary arteries 50 per cent narrowed (2.0 +/- 0.2 versus 2.0 +/- 0.2), near total or total occlusions, coronary score (Friesinger) (7.9 +/- 0.6 versus 8.2 +/- 0.7), left ventricular ejection fraction (48 +/- 2 versus 53 +/- 4), or per cent of akinetic-dyskinetic myocardial segments (66 of 242 [27 per cent] versus 32 of 132 [24 per cent]) between two groups. The similar extent of coronary artery narrowing and degree of left ventricular dysfunction may explain the similar prognosis for patients with transmural myocardial infarction and those with nontransmural myocardial infarction despite differences in enzymatically estimated acute infarct size.

Left ventricular and coronary angiographic anatomy. Relationship to ventricular irritability in the late hospital phase of acute myocardial infarction.

Late hospital phase ventricular arrhythmias in acute myocardial infarction (MI) have been associated with a high incidence of sudden death following hospital discharge. Thirty-eight patients were studied 10-24 days following onset of symptoms of MI. Each patient had a 24-hour ambulatory ECG tape recording and left ventricular and coronary angiography performed. Patients with complicated ventricular arrhythmias (multiform, coupled, R on T VPCs or ventricular tachycardia), when compared to those with uncomplicated ventricular arrhythmias (unifocal or no VPCs), had a greater number of proximally narrowed major coronary arteries (P less than 0.001), a higher coronary "score" (P less than 0.001), a greater incidence of previous myocardial infarction (P less than 0.005), a greater percentage of abnormal left ventricular segments 86% vs 69% (P less than 0.001) and lower ejection fractions. These data suggest that late hospital phase survivors of MI with complicated ventricular arrhythmias have more extensive coronary artery disease with greater left ventricular dysfunction than survivors with uncomplicated ventricular arrhythmias. This more extensive disease may result in increased areas of ischemic myocardium and may help explain the refractoriness of these arrhythmias to pharmacologic therapy.

Sudden death in the year following myocardial infarction. Relation to ventricular premature contractions in the late hospitals phase and left ventricular ejection fraction.

Both depressed left ventricular ejection fraction and ventricular arrhythmias have been associated with a poor prognosis following acute myocardial infarction. To assess the relative role of each of these parameters in predicting mortality in the early period after hospitalization for myocardial infarction, 24 hour ambulatory electrocardiographic tape recordings and gated cardiac blood pool scans were obtained in 81 patients approximately two weeks after their admission to the hospital for myocardial infarction. Lown class 0 to II ventricular premature contractions during this period were classified as uncomplicated ventricular arrhythmias and Lown class III to V ventricular premature contractions were classified as complicated ventricular arrhythmias. Ejection fraction was calculated from biplane images of gated cardiac blood pool scans. In 35 patients the ejection fraction was greater than or equal to 0.40; only three of these had complicated ventricular arrhythmias. In 45 patients the ejection fraction was less than 0.40; 26 of these had complicated ventricular arrhythmias. Eight patients had documented ventricular fibrillation or instantaneous death during a mean 7.0 moonth (range 2 to 16 months) follow-up period outside the hospital. Although the number of patients studied was small, and there were only eight sudden deaths, life table analysis projected a one year mortality of 66 per cent in patients with complicated ventricular arrhythmias and 31 per cent in patients with an ejection fraction less than 0.40. All eight patients who died suddenly were in the subgroup of 26 patients with an ejection fraction less than 0.40 and complicated ventricular arrhymias; none was in the subgroup of 19 patients with an ejection fraction less than 0.40 and uncomplicated ventricular arrhythmias (P less than 0.02). Although a low ejection fraction may suggest a poor prognosis following myocardial infarction, the presence of complicated ventricular arrhythmias significantly increases the risk of sudden cardiac death in the early period after hospitalization in patients with low ejection fraction.

Ventricular arrhythmias in the late hospital phase of acute myocardial infarction. Relation to left ventricular function detected by gated cardiac blood pool scanning.

Abnormalities of left ventricular function and extent of myocardial infarction were studied in relation to prevalence of late ventricular premature contractions (VPCs) in 36 patients in the convalescent stage of acute myocardial infarction (MI). Left ventricular ejection fraction (EF) and percent akinesis (%A) were calculated from gated cardiac blood pool scans; myocardial infarct size was estimated from peak CPK values; and VPCs were detected by 24 hour ambulatory ECGs 2-4 weeks following hospitalization for acute MI. Twenty-two patients had either zero (class 0) or less than 30/hour unifocal VPCs (class I). Fourteen patients had greater than 30/hour unifocal (class II), multifocal (class III) or coupled VPCs (class IV), including ventricular tachycardia. Thirteen of 14 class II-IV patients had EF less than 40% compared with only 8 of 22 class 0-I patients. Class II-IV patients had significantly lower mean EF (30.5 +/- 2.3 SE to 49.6 +/- 4.0) P less than 0.01, higher mean %A (28.1 +/- 2.2 to 16.9 +/- 3.7) P less than 0.05, and higher mean peak CPK (1350 +/- 187 to 721 +/- 155) P less than 0.05 than class 0-I patients. These data suggest that VPCs may not be an independent risk factor for sudden cardiac death in the convalescent phase of MI.