Cigarette smoke components are not very effective in directly inactivating alpha 1-proteinase inhibitor

Cigarette smoke was found to be rather ineffective in inactivating alpha 1-proteinase inhibitor (alpha 1-PI) in aqueous solution, whereas a slow inactivation of alpha 1-PI by a dimethyl sulfoxide extract of whole cigarette smoke condensate was observed. However, this inactivation could only partially be prevented by antioxidants indicating that it is not, or at least not exclusively, due to oxidation. The bulk of inactive alpha 1-PI found in lung lavage fluids from smokers is thus probably generated through endogenous mechanisms and not through smoke components directly.     

Mitochondrial creatine kinase from cardiac muscle and brain are two distinct isoenzymes but both form octameric molecules

Mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle and brain, recently shown to differ in their N-terminal amino acid sequences and to be encoded by multiple mRNAs (Hossle, H.P., Schlegel, J., Wegmann, G., Wyss, M., B?hlen, P., Eppenberger, H. M., Wallimann, T., and Perriard, J.C. (1988) Biochim. Biophys. Res. Commun. 151, 408-416) were separated on two-dimensional nonequilibrium pH-gradient electrophoresis gels and visualized as two distinct protein spots by immunoblotting. Analysis of the two proteins purified by specific elution from Blue-Sepharose with ADP (Wallimann, T., Zurbriggen, B., and Eppenberger, H. M. (1985) Enzyme 33, 226-231) followed by fast protein liquid chromatography cation exchange chromatography showed obvious differences in peptide maps, in immunological cross-reactivity with monoclonal antibodies, and in kinetic parameters. However, even though the two proteins were different, tissue-specific mitochondrial isoforms, both formed regularly-sized, perforated cube-like octameric structures with Mr of 364,000 +/- 25,000 and 352,000 +/- 20,000 for the cardiac and brain isoform, respectively. Electron microscopy of cardiac and brain Mi-CK octamers revealed cube-like molecules with a central cavity or transverse channel filled by negative stain. The octameric molecular structure of Mi-CK isoforms differs from the generally accepted dimeric arrangement of "cytosolic" muscle MM- and brain BB-CK.     

Enzymatic oxidation of p-nitrophenol.

A Moraxella sp. capable of growth with p-nitrophenol was isolated from activated sludge. Differential centrifugation of crude cell extracts gave a membrane preparation that oxidized p-nitrophenol to hydroquinone and nitrite. Enzymatic activity was dependent on the presence of oxygen and reduced pyridine nucleotides and was stimulated by the addition of flavin adenine dinucleotide. Experiments with ¹⁸O₂ showed that the incoming hydroxyl group was derived from molecular oxygen. The soluble fraction prepared from crude cell extracts oxidized hydroquinone to a product whose spectral properties were identical to those reported for γ-hydroxymuconic semialdehyde.     

High resolution respirometry of permeabilized skeletal muscle fibers in the diagnosis of neuromuscular disorders

High resolution respirometry in combination with the skinned fiber technique offers the possibility to study mitochondrial function routinely in small amounts of human muscle. During a period of 2 years, we investigated mitochondrial function in skeletal muscle tissue of 13 patients (average age = 5.8 years). In all of them, an open muscle biopsy was performed for diagnosis of their neuromuscular disorder. Mitochondrial oxidation rates were measured with a highly sensitive respirometer. Multiple substrate-inhibitor titration was applied for investigation of mitochondrial function. About 50 mg fibers were sufficient to obtain maximal respiratory rates for seven different substrates (pyruvate/malate, glutamate/malate, octanoylcarnitine/malate, palmitoylcarnitine /malate, succinate, durochinol and ascorbate/TMPD). Decreased respiration rates with reference to the wet weight of the permeabilized fiber could immediately be detected during the course of measurements.In 4 patients with mitochondrial encephalomyopathy (MEM) the respiration pattern indicated a specific mitochondrial enzyme defect, which was confirmed in every patient by measurements of the individual enzymes (one patient with PDHC deficiency, one with complex I deficiency and two patients with combined complex I and IV deficiency). In the 6 patients with spinal muscular atrophy (SMA) oxidation rates were found to be decreased to 23 ± 5% of controls. The normalized respiration pattern was comparable to that of the controls indicating a decreased content of mitochondria in SMA muscle with normal functional properties. Also in the 3 patients with Duchenne muscular dystrophy (DMD) decreased oxidation rates (42 ± 5%) were detected. In addition a low RCI (1.2) indicated a loose coupling of oxidative phosphorylation in the mitochondria of these patients.It is concluded that investigation of mitochondrial function in saponin skinned muscle fibers using high resolution respirometry in combination with multiple substrate titration offers a valuable tool for evaluation of mitochondrial alterations in muscle biopsies of children suffering from neuromuscular disorders. (Mol Cell Biochem 174: 71–78, 1997)     

Modification of different IgG1 antibodies via glutamine and lysine using bacterial and human tissue transglutaminase

The modification of proteins by chemical methods is well-established, however usually difficult to control. In this paper, we describe the posttranslational modification of different IgGs via the Lys or Gln side chains catalyzed by bacterial and human tissue transglutaminase (BTGase and TG2). For proof of concept, different IgG1s (commercial bovine IgG1, and L1CAM targeting chCE7 and chCE7 aglycosylated) were enzymatically functionalization with different fluorescent TGase substrates based on the CY3 analogue Dy547. The optimal reaction conditions were determined in order to assess the two enzymes. The efficiency of the enzymatic method was also compared with a standard chemical method employing a reactive NHS ester of Dy547. Three new TGase substrates were synthesized for this study including Lys-substrate 1 useful for BTGase and TG2 and two Gln-substrates tailor-made for BTGase (substrate 2) and TG2 (substrate 3). Of the two TGases tested, BTGase incorporated Lys-substrate 1 more efficiently than TG2. On the other hand, both enzymes reacted equally efficiently with the corresponding Gln-substrates 2 and 3. Reproducible labeling could be achieved in a broad concentration "window" of the substrates (up to 400 microM) without the risk of overlabeling of chCE7 or chCE7 aglycosylated. The biological activities of the functionalized antibodies were unaltered as shown by in vitro antigen affinity measurements and cell internalization experiments using confocal laser scanning microscopy. A maximum label-to-protein ratio of approximately 1 was achieved with chCE7 aglycosylated and substrate 1 using BTGase. It is important to recognize that the enzymatic activity of TGases enables the stable functionalization of proteins via the side chains of Gln, which is not possible by any chemical method available today. In addition, we could prove that the enzymatic modification of all antibodies occurred selectively at the heavy chain whereas the chemical method led to labeling of both the heavy and the light chains.     

Functional studies with the octameric and dimeric form of mitochondrial creatine kinase. Differential pH-dependent association of the two oligomeric forms with the inner mitochondrial membrane

Phosphate extraction of mitochondrial creatine kinase (Mi-CK, EC 2.7.3.2) from freshly isolated intact mitochondria of chicken cardiac muscle, after short swelling in hypotonic medium, yielded more than 90% of octameric and only small amounts of dimeric Mi-CK as judged by fast protein liquid chromatography-gel permeation analysis of the supernatants immediately after extraction of the enzyme. In extraction buffer, octameric Mi-CK displayed a tendency to dissociate, albeit at a slow rate with a half-life of approximately 3-5 days, into stable dimers. Experiments with purified Mi-CK octamers or dimers, or defined mixtures thereof, incubated under identical conditions with Mi-CK-depleted mitoplasts revealed that both oligomeric forms of Mi-CK can rebind to mitoplasts. However, the association of Mi-CK was strongly pH-dependent and, in addition, octameric and dimeric Mi-CK showed different pH dependences of rebinding. Therefore, it was possible under certain pH conditions to rebind either both oligomeric forms or selectively the octamers only. Furthermore, evidence is presented that Mi-CK dimers partially form octamers upon rebinding to the inner membrane. The differential association of the two oligomeric Mi-CK forms with the inner mitochondrial membrane together with the dynamic equilibrium between octameric and dimeric Mi-CK (Schlegel, J., Zurbriggen, B., Wegmann, G., Wyss, M., Eppenberger, H.M., and Wallimann, T. (1988) J. Biol. Chem., 263, 16942-16953) suggest that both oligomeric forms are physiologically relevant. A change in the octamer to dimer ratio may influence the association behavior of Mi-CK in general and thus modulate mitochondrial energy flux as discussed in the phosphoryl creatine circuit model (Wallimann, T., Schnyder, T., Schlegel, J., Wyss, M., Wegmann, G., Rossi, A.-M., Hemmer, W., Eppenberger, H.M., and Quest, A.F.G. (1989) Prog. Clin. Biol. Res. 315, 159-176.