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ABSTRACT: During recent years the dual role of endogenous inflammatory mediators such as tumour necrosis factor (TNF) has become evident. While TNF has been recognised to possess a great detrimental potential, for example in the case of sepsis, it is on the other hand an integral component of an adequate immune response to bacterial invasion. These different properties of TNF and others seem to be dependent mainly on the quantitative extent of their formation. Some recent findings indicate that this extent may in part be determined genetically. The classification of patients according to polymorphic cytokine genes might, therefore, predict some of their reactions to septic challenges.
The European journal of surgery. Supplement.: = Acta chirurgica. Supplement 02/1999;
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ABSTRACT: The cytokine production in endotoxin stimulated blood of patients immediately after polytrauma with high risk for developing sepsis or multi organ failure was analysed. Forty patients sustaining traumatic injury with >/=317 pts according to the Injury Severity Score (ISS), 10 of whom developed severe sepsis (ACCP/SCCM conference 1992) were included in the study. Levels of interleukin 8 (IL-8), IL-6 and tumour necrosis factor (TNF) were measured by ELISA in endotoxin-stimulated whole blood and IL-10 and IL-6 in serum. The allotype for the bi-allelic Nco I restriction length polymorphism in the TNF locus was determined for each patient.Two to four hours after polytrauma endotoxin-stimulated synthesis of TNF and IL-6 was found to be reduced in whole blood from patients compared to healthy donors, whereas no such differences were found for IL-8 synthesis. At this time, however, the patients who developed sepsis at a later stage (day 4-6) showed significantly (P<0.05) enhanced IL-8 synthesis in endotoxin stimulated whole blood in comparison to healthy donors. The IL-6 and TNF production of their blood was significantly enhanced compared to patients with uncomplicated recovery. Ninety per cent of the patients developing sepsis were of the TNFB2/TNFB2 allotype, whereas this was the case for only 30% of the non-septic group. Assessment of endotoxin-stimulated cytokine synthesis may provide a prognostic indicator for patients at high risk for developing a sepsis syndrome.
Cytokine 02/1999; 11(2):173-8. · 3.02 Impact Factor
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ABSTRACT: The benefit of early operative stabilization of femoral fractures is established in patients with multiple injuries. In the last few years the unreamed femoral nail is favoured for internal fixation of femoral fractures despite pathophysiological concerns. The foremost advantage of femoral nails compared with plate fixation is the possibility of early full weight bearing. The aim of this retrospective study was to investigate, under consideration of the severity of injury, the extent of injury, and the clinical course, if multiple injured patients with concomitant femoral fractures benefit from the preferred intramedullary nailing with early weight bearing. Three hundred and two (23.8%) out of 1271 multiple injured patients (ISS > 17) had a concomitant femoral fracture. Fourty-seven out of 302 patients were children under 16 years of age, remaining 255 patients. Eighteen out of 255 patients died within the first 21 days after trauma and 66 patients required mechanical ventilation for more than three weeks (171/255). Thirty patients suffered from severe head injury (AIS-head > 3) and seven from severe pulmonary contusion with concomitant abdominal injury (134/255). Two patients had grade III open femoral fractures with vascular injury. Ipsilateral unstable pelvic fractures were seen in 11 patients, seven patients had ipsilateral intraarticular femoral fractures, and ipsilateral intraarticular fractures of the lower leg or foot were observed in 40 patients (74/255). The results demonstrate, that only 74 (29%) out of 255 multiple injured patients (> 16 years of age) had a theoretical benefit of early weight bearing. Seventy percent of the patients did not benefit from intramedullary nailing considering full weight bearing. With regard to pathophysiological concerns alternative methods of fracture fixation should be discussed for these patients. Primary fracture fixation with external fixators and secondary internal fixation proved to be a save alternative method. The complication rate of plating is comparable to intramedullary nailing but associated with less severe systemic risks. Primary plating of femoral fractures would not delay mobilization of most multiple injured patients.
Der Unfallchirurg 11/1998; 101(10):769-74. · 0.61 Impact Factor
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U Obertacke,
F Neudeck, M Majetschak,
A Hellinger,
C Kleinschmidt,
F U Schade,
K Høgasen,
M Jochum,
W Strohmeier,
M Thurnher,
H Redl,
G Schlag
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ABSTRACT: The present study was designed to investigate the consequences of isolated unilateral lung contusion on local alveolar and systemic inflammatory responses in an animal model in the pig. Isolated unilateral lung contusion was induced by bolt shot in eight mechanically ventilated animals under general anesthesia (sham: n=4). Plasma and bronchoalveolar lavage fluid were collected during a period of 8 h following lung contusion. Leukocytes, leukocyte neutral protease inhibitor (LNPI), terminal complement complex (TCC), thrombin-antithrombin-complex (TAT) as well as pulmonary microvascular permeability and surfactant function were determined. Within 30 min, lung contusion was found to cause a significant local and systemic increase in TCC and TAT concentrations and a systemic increase in LNPI concentrations. The latter was accompanied by a sequestration of leukocytes in the contused lung. Complement activation and leukocyte sequestration in the contused lung progressively increased during the investigation period. Although surfactant function decreased in the entire lung 30 min after contusion, TCC, TAT, and leukocyte sequestration was unchanged in the contralateral lung. The first indication of an involvement of the contralateral lung was obtained by an increase in leukocyte sequestration 8 h after lung contusion. Unilateral lung contusion initiates an early systemic activation of humoral and cellular defense systems. Involvement of the contralateral lung appears to be a secondary event caused by a systemic inflammatory reaction.
Shock 08/1998; 10(1):7-12. · 2.85 Impact Factor
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ABSTRACT: Ubiquitin is often implicated as a specific tag for protein degradation via the ubiquitin system although only a limited number of physiological proteins have been shown to be degraded in their native tissues via this pathway in vivo. Ubiquitin may also, however, have other functions of a regulatory nature (non-catabolic ubiquitylation). The ubiquitylation of calmodulin appears to fall into this category. Ubiquitin is linked to free calmodulin in the presence of the second messenger Ca2+ by the enzyme ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21) and there is no evidence that this step is followed by degradation of calmodulin via the ATP-dependent 26-S protease. Due to a lack of natural substrates and sufficient tissue material, only a few components of the ubiquitin system have been obtained in truly homogeneous form from reticulocytes. We therefore decided to attempt this for the calmodulin ligase. The enzymic components of the uCaM synthetase system copurified over several steps and could be highly enriched by a novel sample displacement technique on an ion-exchange resin. A fractionation of the synthetase components by affinity chromatography on ubiquitin-Sepharose and calmodulin-Sepharose yielded two essentially inactive components: a ubiquitin-Sepharose binding fraction (uCaM Syn-F1) and a calmodulin-Sepharose binding fraction (uCaM Syn-F2). The full activity of uCaM synthetase can be reconstituted when these two fractions are reunited. uCaM Syn-F1 could then be separated from all other enzymes of ubiquitin metabolism and, employing the second component with the natural substrate calmodulin, could be purified over 3500-fold to homogeneity. The ability to catalyze its own thiol labile ubiquitylation identified it as a member of the ubiquitin-activating enzyme family (E1). The homogeneous preparation contained a single protein of molecular mass 213 +/- 21 kDa (mean +/- SEM) as determined by gel filtration. The molecular mass of the monomer was determined by electrospray ion mass spectrometry to 112,140 +/- 47 Da (mean +/- SD). N-terminal sequence analysis (20 amino acids) led to a single N-terminal peptide beginning at residue 57 of the known rabbit cDNA sequence. No ragged N-terminus was detected, as would be expected by the action of an aminopeptidase or other peptidases of low specificity. The monomer molecular mass calculated from the cDNA sequence (Arg57-Arg1058) is 111,975 Da, characterizing this enzyme from reticulocytes as a homodimer of 224 kDa.
European Journal of Biochemistry 08/1998; 255(2):482-91. · 3.58 Impact Factor
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ABSTRACT: Ubiquitin-calmodulin ligase (uCaM synthetase: EC 6.3.2.21), which has been detected in all tissues so far examined, catalyzes the Ca2+-dependent reversible synthesis of ubiquityl-calmodulin which is not directed to degradation by the ATP-dependent 26-S protease [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173-191]. As has been shown in the preceding paper in this journal, the uCaM synthetase holosystem can be separated into two essential protein components: uCaM Syn-F1, a ubiquitin-binding protein belonging to the ubiquitin-activating enzyme family (E1) and uCaM Syn-F2 which bestows the reaction specificity leading to the covalent modification of calmodulin with ubiquitin. UCaM Syn-F2, which binds to calmodulin-Sepharose in a Ca2+-dependent manner, has been purified over 3500-fold in seven steps from rabbit reticulocytes and has a native molecular mass of approximately 620 kDa. It binds calmodulin with a Km of 5 microM and to uCaM Syn-F1, i.e. ubiquitin-activating enzyme (E1), with a Km of 3 nM. The maximal specific activity obtained in enriched uCaM Syn-F2 is 6-8 pkat/mg. The pH optimum of uCaM synthetase lies at pH 8.5. In kinetic experiments the Km values for 125I-ubiquitin and ATP/Mg2+ were determined to be 8 microM and 16 nM, respectively, for the uCaM synthetase holosystem. The existence of a third separable protein component of uCaM synthetase, as is the case in E1, E2, E3 systems, is very unlikely since affinity chromatography on calmodulin-Sepharose, two ion-exchange chromatography steps and finally a gel-filtration step failed to indicate any additional protein component essential for synthetase activity. We therefore propose a two-component model for uCaM synthetase. This model is also supported by simple hyperbolic velocity curves in kinetic experiments based on the variation of these two components. The data suggests that uCaM Syn-F2 is neither an E2 nor an E3 but evidently combines the properties of both, making the Ca2+-dependent uCaM synthetase the member of a group of two-component ubiquitin ligase systems.
European Journal of Biochemistry 07/1998; 255(2):492-500. · 3.58 Impact Factor
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ABSTRACT: The benefit of early operative stabilization of femoral fractures is established in patients with multiple injuries. In the
last few years the unreamed femoral nail is favoured for internal fixation of femoral fractures despite pathophysiological
concerns. The foremost advantage of femoral nails compared with plate fixation is the possibility of early full weight bearing.
The aim of this retrospective study was to investigate, under consideration of the severity of injury, the extent of injury,
and the clinical course, if multiple injured patients with concomitant femoral fractures benefit from the preferred intramedullary
nailing with early weight bearing. Three hundred and two (23.8 %) out of 1271 multiple injured patients (ISS > 17) had a concomitant
femoral fracture. Fourty-seven out of 302 patients were children under 16 years of age, remaining 255 patients. Eighteen out
of 255 patients died within the first 21 days after trauma and 66 patients required mechanical ventilation for more than three
weeks (171/255). Thirty patients suffered from severe head injury (AIS-head > 3) and seven from severe pulmonary contusion
with concomitant abdominal injury (134/255). Two patients had grade III open femoral fractures with vascular injury. Ipsilateral
unstable pelvic fratures were seen in 11 patients, seven patients had ipsilateral intraarticular femoral fractures, and ipsilateral
intraarticular fractures of the lower leg or foot were observed in 40 patients (74/255). The results demonstrate, that only
74 (29 %) out of 255 multiple injured patiens ( > 16 years of age) had a theoretical benefit of early weight bearing. Seventy
percent of the patients did not benefit from intramedullary nailing considering full weight bearing. With regard to pathophysiological
concerns alternative methods of fracture fixation should be discussed for these patients. Primary fracture fixation with external
fixators and secondary internal fixation proved to be a save alternative method. The complication rate of plating is comparable
to intramedullary nailing but associated with less severe systemic risks. Primary plating of femoral fractures would not delay
mobilization of most multiple injured patients.
Der Vorteil der primären operativen Stabilisierung von Femurfrakturen beim Polytraumatisierten ist anerkannt. Trotz grundsätzlicher
pathophysiologisch begründeter Einwände gegen einen intramedullären Kraftträger zur primären Stabilisierung der Oberschenkelfraktur
wird in den letzten Jahren zunehmend der ungebohrte Marknagel favorisiert. Zentrales Argument der Befürworter des Marknagels
ist die Frühbelastbarkeit. In der vorliegenden retrospektiven Studie wurde untersucht, wieviele Polytraumatisierte mit Femurfraktur
unter Berücksichtigung der Verletzungsschwere, des Verletzungsmusters und des klinischen Verlaufes den Vorteil der Frühbelastung
nach primärer Marknagelung hätten nutzen können. Von 1271 polytraumatisierten Patienten (PT) (ISS > 17 Punkte) hatten 302
(23,8 %) eine Femurfraktur, davon 47 Kinder < 16 Jahre, so daß 255 PT zur Frage der primären Stabilisierungsform verblieben.
Von den 255 PT (100 %) verstarben 18 PT innerhalb der ersten 21 Tage und 66 PT mußten länger als 21 Tage auf der Intensivstation
behandelt werden (171/255); 30 PT erlitten ein Schädel-Hirn-Trauma III° und 7 eine schwere Lungenkontusion mit begleitender
Abdominalverletzung (134/255); 2 PT hatten III° offene Femurfrakturen mit Gefäßrekonstruktion. Ipsilaterale Beckenverletzungen
hatten 11 PT, 7 PT Gelenk- bzw. gelenknahe Frakturen des gleichen Femurs und 40 PT hatten ipsilaterale Gelenkfrakturen am
Unterschenkel bzw. Fuß (74/255). Retrospektiv hätten von 255 PT > 16 Jahre mit Femurfraktur nur 74 (29 %) die Möglichkeit
der Frühbelastung einer Marknagelung nutzen können. Da mehr als 70 % der PT die Marknagelung einer Femurfraktur unter dem
Aspekt „Frühbelastung“ nicht hätten nutzen können, stellt sich unter Zugrundelegung pathophysiologischer Bedenken die Frage,
ob nicht für diese Patienten alternative Stabilisierungsformen gewählt werden sollten. Für schwerstmehrfachverletzte Patienten
steht primär der Fixateur externe mit sekundär möglichem Verfahrenswechsel zur Verfügung. Für einen großen Teil der Polytraumatisierten
würde eine primäre Plattenosteosynthese der Femurfraktur bei geringerem systemischen Risiko und gleicher Komplikationsrate
die Mobilisierung nicht verzögern.
Der Unfallchirurg 04/1998; 101(10):769-774. · 0.61 Impact Factor
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ABSTRACT: Cardiac surgery and polytrauma result in an impaired immune response as it can be demonstrated by a reduced endotoxin-stimulated TNF alpha production of whole blood cultures ex vivo. The immune-stimulating hematopoetic growth factor GM-CSF is in vitro capable to antagonize the suppressed immune function after trauma and cardiac surgery and, therefore, GM-CSF represents a potential therapeutic for immune suppressed states.
Langenbecks Archiv für Chirurgie. Supplement. Kongressband. Deutsche Gesellschaft für Chirurgie. Kongress 02/1998; 115(Suppl I):169-72.
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ABSTRACT: Trauma has been recognized to be accompanied by alterations of leukocyte functions such as cytokine release. The regulatory principles involved in these changes are still poorly defined. To further characterize leukocyte function after multiple trauma, endotoxin-stimulated tumor necrosis factor (TNF) production of trauma patients' whole blood and a possible regulatory mechanism were studied.
Endotoxin responsiveness in trauma patients (n = 18, Injury Severity Score = 24 +/- 7) was assayed ex vivo using a whole blood model. TNF release and TNFalpha mRNA levels were determined during a 14-day period. Furthermore, the influence of patients' sera on whole blood TNF production was evaluated.
The capacity of trauma patients' whole blood to produce TNF was reduced for 2 to 6 days after trauma and was equally evident for both TNF release and TNFalpha mRNA levels. The reduction of TNF coincides with the appearance of an inhibitory activity for TNF production in trauma patients' sera. No correlation was found between the inhibitory activity and soluble TNF receptors, endotoxin-neutralizing molecules, inhibitory cytokines (interleukin 10 and transforming growth factor beta), or prostaglandins.
Major trauma leads to the appearance of a circulating inhibitory activity for TNF synthesis that may potentially contribute to an anti-inflammatory response in patients with multiple trauma. The elucidation of its structural and functional properties may contribute to the understanding of the pathogenesis of severely injured patients.
The Journal of trauma 01/1998; 43(6):880-7. · 2.48 Impact Factor
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ABSTRACT: Accident proceedings and injuries were investigated in a consecutive series of 70 patients (65.2% male, 34.8% female, age: 18 +/- 9 years) presented to the emergency department after in-line skate falls. 52.7% wore no protective equipment. 41.8% used knee pads, 27.3% wrist guards, 12.7% elbow pads, and 5.5% each helmets and gloves. The most common cause of fall were obstacles/irregularities (32.7%) on a dry and plain surface. The upper extremity (67.9%) and the head (12.8%) were the most commonly injured body parts. 47.8% in-line skaters sustained fractures. The distal radius (51.5%) was most commonly injured body parts. 47.8% in-line skaters sustained fractures. The distal radius (51.5%) was most commonly fractured. Injuries to novice skaters were more often severe. Advanced freestyle and jumping in-line skaters sustained even injuries of the trunk. Life-threatening injuries of the abdomen and the head may occur. The use of a complete personal protection equipment is recommended.
Unfallchirurgie 09/1997; 23(4):171-8.
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ABSTRACT: Pulmonary gas exchange in correlation with condensed lung volume was prospectively studied in 10 patients with multiple injuries and blunt chest trauma. The purpose was to find nomograms that allow the estimation of the extent of pulmonary density from gas exchange parameters. The condensed lung volume was determined planimetrically from serial transverse sections of chest computed tomographic scans. There was no correlation between condensed lung volume and mean pulmonary artery pressure, pulmonary vascular resistance, systemic vascular resistance, or cardiac index and a week negative correlation to the oxygenation index (PaO2/FIO2) (r2 = 0.46) and to the total static lung compliance (r2 = 0.29). A strong correlation between pulmonary density and intrapulmonary shunt fraction (Qs/Qt) (r2 = 0.95) as well as alveoloarterial PO2 difference (P[A-a]O2) (r2 = 0.86) was evident. By using linear regression equations (linear regression line with 95% confidence interval), nomograms were calculated. The extent of pulmonary density can easily be obtained from these nomograms by measuring Qs/Qt or P(A-a)O2. The presented nomograms may be helpful in monitoring the effect of treatment in patients with blunt chest trauma.
The Journal of trauma 08/1997; 43(1):8-12. · 2.48 Impact Factor
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ABSTRACT: Multiple injuries in elderly patients are still a common problem. The present study was performed to investigate mortality and complications in multiple trauma patients aged 65 years or more. A total of 1154 multiple trauma patients with an injury severity score (ISS) of at least 18 points were divided in two age groups: Y: 16-64 years, n = 1022; O: 65-94 years, n = 132. Older patients were injured as pedestrians in most cases (69%), while younger patients were more frequently injured as car and drivers passengers (41%). ISS was comparable in both groups (Y 28 +/- 1, O 27 +/- 1). During ICU-therapy incidence of ARDS (Y 10%, O 11%), multiple organ dysfunction syndrome (MOF; Y 6%, O 9%) and pneumonia (Y 17%, O 21%) were comparable. In contrast, septic complications were more frequent in older patients (Y 19%, O 27%). Length of ICU stay (Y 19 +/- 2, O 18 +/- 1) and ventilation time (Y 14 +/- 2, O 17 +/- 1) were comparable. Mortality was significantly higher in older patients (Y 15%, O 53%). The major cause of death was sepsis in older patients (Y 15%, O 31%) and MOF in younger patients (Y 54%, O 29%). In conclusion, older trauma patients had a higher mortality due to the development of septical complications.
Der Unfallchirurg 07/1997; 100(6):477-82. · 0.61 Impact Factor
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ABSTRACT: In einer prospektiven klinischen Studie wurden Unfallmechanismen und Verletzungsmuster bei 70 konsekutiven In-line-Skating-Unfällen
(65,2% männlich, 34,8% weiblich, Alter: 18±9 Jahre) untersucht. 52,7% der Patienten fuhren ohne jede Schutzkleidung, 41,8%
trugen Knieschoner, 27,3% Handgelenkprotektoren, 12,7% Ellenbogenschoner und je 5,5% Helme bzw. Handschuhe. Die häufigsten
Unfallursachen waren kleine Hindernisse (32,7%) auf trockener und ebener Fahrbahn. Am häufigsten war die obere Extremität
verletzt (67,9%), gefolgt von Verletzungen des Kopfes (12,8%). Frakturen wurden bei 47,8% der In-line-Skater festgestellt.
Die häufigste Fraktur war die distale Radiusfraktur (51,5%). Den höchsten Anteil schwerer Verletzungen wiesen Anfänger auf.
Bei Verletzungen nach Sprüngen mit In-line-Skates traten auch schwere stammnahe Verletzungen auf. Mit lebensbedrohlichen Verletzungen
innerer Organe sowie schweren Schädel-Hirn-Verletzungen muß gerechnet werden. Die Anwendung einer kompletten Schutzausrüstung
wird empfohlen.
Accident proceedings and injuries were investigated in a consecutive series of 70 patients (65.2% male, 34.8% female, age:
18±9 years) presented to the emergency department after in-line skate falls. 52.7% wore no protective equipment. 41.8% used
knee pads, 27.3% wrist guards, 12.7% elbow pads, and 5.5% each helmets and gloves. The most common cause of fall were obstacles/irregularities
(32.7%) on a dry and plain surface. The upper extremity (67.9%) and the head (12.8%) were the most commonly injured body parts.
47.8% in-line skaters sustained fractures. The distal radius (51.5%) was most commonly fractured. Injuries to novice skaters
were more often severe. Advanced freestyle and jumping in-line skaters sustained even injuries of the trunk. Life-threatening
injuries of the abdomen and the head may occur. The use of a complete personal protection equipment is recommended.
European Journal of Trauma 04/1997; 23(4):171-178.
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ABSTRACT: Die Versorgung polytraumatisierter, älterer Patienten ist aufgrund vielfältiger Probleme besonders aufwendig und mit einer
hohen Letalität bei diesen Patienten verknüpft. Der Verlauf und die Prognose älterer polytraumatisierter Patienten sollte
durch Vergleich mit jüngeren Patienten untersucht werden. Dazu wurden 2 Gruppen gebildet: Gruppe J: 16 – 64 Jahre, n = 1022, Gruppe A: 65 – 94 Jahre, n = 132. Ältere Patienten wurden häufiger als Fußgänger (69%), jüngere Patienten vor allem als PKW-Insassen (41%) verletzt.
Die Verletzungsschwere im ISS (J: 28±1, A: 27±1) war in beiden Gruppen gleich. Im Verlauf traten ARDS (J: 10%, A: 11%), MOV
(J: 6%, A: 9%) und Pneumonien (J: 17%; A: 21%) in beiden Gruppen gleichhäufig auf, während septische Komplikationen bei den
älteren Patienten signifikant häufiger beobachtet wurden (J: 19%, A: 27%). Ältere Patienten verstarben signifikant häufiger
(J: 16%, A: 58%) an der Sepsis (J: 15%, A: 31%), jüngere Patienten dagegen häufiger an den Folgen des Organversagens (J: 54%,
A: 29%). Die Intensiv-(J: 19±2, A: 18±1) und Beatmungsdauer (J: 17±1, A: 14±2) war bei den älteren Patienten nicht verlängert.
Zusammengefaßt haben ältere Patienten nach schweren Unfallverletzungen bei vergleichbarer anatomischer Verletzungsschwere
eine deutlich höhere Letalität als jüngere Patienten, wobei diese auf der Entwicklung septischer Komplikationen beruht.
Multiple injuries in elderly patients are still a common problem. The present study was performed to investigate mortality
and complications in multiple trauma patients aged 65 years or more. A total of 1154 multiple trauma patients with an injury
severity score (ISS) of at least 18 points were divided in two age groups: Y: 16 – 64 years, n = 1022; O: 65 – 94 years, n = 132. Older patients were injured as pedestrians in most cases (69%), while younger patients were more frequently injured
as car and drivers passengers (41%). ISS was comparable in both groups (Y 28±1, O 27±1). During ICU-therapy incidence of ARDS
(Y 10%, O 11%), multiple organ dysfunction syndrome (MOF; Y 6%, O 9%) and pneumonia (Y 17%, O 21%) were comparable. In contrast,
septic complications were more frequent in older patients (Y 19%, O 27%). Length of ICU stay (Y 19±2, O 18±1) and ventilation
time (Y 14±2, O 17±1) were comparable. Mortality was significantly higher in older patients (Y 15%, O 53%). The major cause
of death was sepsis in older patients (Y 15%, O 31%) and MOF in younger patients (Y 54%, O 29%). In conclusion, older trauma
patients had a higher mortality due to the development of septical complications.
Der Unfallchirurg 04/1997; 100(6):477-482. · 0.61 Impact Factor
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ABSTRACT: Ubiquityl-calmodulin synthetase (uCaM-synthetase) activity as detected in reticulocyte lysate and the crude extracts of rabbit tissues [FEBS Lett. 294 (1991) 229-233] has been well characterized as being essentially Ca(2+)-dependent (-Ca2+/+Ca2+ activity ratio: 0.15-0.2). However, during the purification of this enzyme on ubiquitin-Sepharose the Ca(2+)-dependent activity is lost and an essentially Ca(2+)-independent enzyme (-Ca2+/+Ca2+ activity ratio: 1.0-1.5) is obtained which was purified 90-fold (uCaM-Syn F1) to a final specific activity of 0.32 pkat/mg. During the purification procedure a second protein factor (uCaM-Syn F2) was isolated that has no catalytic activity by itself but restores Ca2+ dependence to the uCaM-Syn F1 fraction (-Ca2+/+Ca2+ activity ratio: 0.1) and enhances the catalytic activity in uCaM-Syn F1 in the presence of Ca2+ over 40-fold. It is concluded that several (possibly interdependent) forms of uCaM-synthetase exist which display different substrate specificities for calmodulin.
FEBS Letters 02/1993; 315(3):347-52. · 3.54 Impact Factor
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ABSTRACT: Recently we were able to show that calmodulin from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner by ubiquityl-calmodulin synthetase (uCaM-synthetase) from mammalian sources [R. Ziegenhagen and H.P. Jennissen (1990) FEBS Lett. 273, 253-256]. It was therefore of high interest to investigate whether this covalent modification of calmodulin also occurs in one of the simplest eukaryotes, the unicellular Saccharomyces cerevisiae. Yeast calmodulin was therefore purified from bakers yeast. In contrast to calmodulin from spinach and N. crassa it does not activate phosphorylase kinase. Crude yeast uCaM-synthetase conjugated ubiquitin Ca(2+)-dependently to yeast and mammalian (bovine) calmodulin. Yeast calmodulin was also a substrate for mammalian (reticulocyte) uCaM-synthetase. As estimated from autoradiograms the monoubiquitination product (first-order conjugate) of yeast calmodulin has an apparent molecular mass of ca. 23-26 kDa and the second-order conjugate an apparent molecular mass of ca. 28-32 kDa. Two to three ubiquitin molecules can be incorporated per yeast calmodulin. Experiments with methylated ubiquitin in the heterologous reticulocyte system indicate that, as with vertebrate calmodulins, only one lysine residue of yeast calmodulin reacts with ubiquitin so that the incorporation of multiple ubiquitin molecules will lead to a polyubiquitin chain. These results also indicate that the ability of coupling ubiquitin to calmodulin was acquired at a very early stage in evolution.
FEBS Letters 02/1992; 296(1):51-6. · 3.54 Impact Factor
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ABSTRACT: Ubiquityl-calmodulin synthetase (uCaM-synthetase) activity as detected in reticulocyte lysate and the crude extracts of rabbit tissues [FEBS Lett. 294 (1991) 229-233] has been well characterized as being essentially Ca2+-dependent (−Ca2+/+Ca2+ activity ratio: 0.15–0.2). However, during the purification of this enzyme on ubiquitin-Sepharose the Ca2+-dependent activity is lost and an essentially Ca2+-independent enzyme (−Ca2++Ca2+ activity ratio: 1.0–1.5) is obtained which was purified 90-fold (uCaM-Syn Fl) to a final specific activity of 0.32 . During the purification procedure a second protein factor (uCaM-Syn F2) was isolated that has no catalytic activity by itself but restores Ca2+ dependence to the uCaM-Syn Fl fraction (−Ca2++Ca2+ activity ratio: 0.1) and enhances the catalytic activity in uCaM-Syn Fl in the presence of Ca2+ over 40-fold. It is concluded that several (possibly interdependent) forms of uCaM-synthetase exist which display different substrate specificities for calmodulin.
FEBS Letters.
