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M Rohde

Publications and source records attributed to M Rohde.

138 records · Page 8Linked to original sources
PubMed1986

Brain pyridoxal kinase. Purification and characterization.

Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure.

Animals↗
PubMed1986

Pyruvate carboxylase from Saccharomyces cerevisiae. Quaternary structure, effects of allosteric ligands and binding of avidin.

The quaternary structure of pyruvate carboxylase purified from Saccharomyces cerevisiae was investigated by electron microscopic examination of negatively stained samples. In the most frequently observed projection form four intensity maxima were arranged at the corners of a rhombus; a cleft along the longitudinal axis of individual protomers could often be discerned. The observation of occasional triangular and dual-intensity projections and the interconversion of all three projection forms in tilting studies indicates that this tetrameric enzyme has a structure very similar to the tetrahedron-like configuration previously proposed for pyruvate carboxylases from vertebrate sources [Mayer, F., Wallace, J. C. and Keech, D. B. (1980) Eur. J. Biochem. 112, 265-272] and Aspergillus nidulans [Osmani, S. A., Mayer, F., Marston, F. A. O., Selmes, I. P. and Scrutton, M. C. (1984) Eur. J. Biochem. 139, 509-518]. An improved structural preservation of the enzyme was observed in the presence of either of the activators acetyl-CoA (250 microM) and palmitoyl-CoA (1-5 microM). At higher than 5 microM palmitoyl-CoA, although activity was further increased, dissociation of enzyme tetramers was evident, presumably because of the detergent effect of the long-chain acyl moiety. Two inhibitors of yeast pyruvate carboxylase, L-aspartate (10 mM) and 2-oxoglutarate (40 mM), added alone or together decreased significantly the proportion of intact tetramers even in the presence of acetyl-CoA or palmitoyl-CoA. When yeast pyruvate carboxylase was incubated with avidin, the formation of unbranched linear concatemers occurred at avidin:enzyme ratios between 2:1 and 1:2. Avidin molecules were sometimes bound asymmetrically to the enzyme, appearing to complex only one biotin group on each side of the enzyme. This appeared to permit kinking and circularization of some concatemers.

Allosteric Site↗
PubMed1986

Clobazam for refractory focal epilepsy. A controlled trial.

The effect of 1,5-benzodiazepine clobazam was assessed in a double-blind add-on trial in 20 patients with chronic complex partial seizures uncontrolled by maximally tolerable daily dosage of standard antiepileptic drug therapy. The number of seizures was lower during the three months of active treatment. At the end of the third month, eight (40%) of the patients had a seizure reduction by more than 75%, including four patients (20%) who had complete control. Tolerance to the antiepileptic effect of clobazam was noted in 56% of the patients, and mild transient sedation occurred in 40% of the patients. Despite these drawbacks, clobazam is an effective add-on drug for individual patients with refractory focal epilepsy.

Adolescent↗
PubMed1984

Immunocytochemical localization of carbon monoxide oxidase in Pseudomonas carboxydovorans. The enzyme is attached to the inner aspect of the cytoplasmic membrane.

The localization of carbon monoxide oxidase (CO oxidase), the key enzyme in CO metabolism of Pseudomonas carboxydovorans, was examined using modified immunoferritin and protein A-gold techniques. Cell extracts were incubated with specific immunoglobulin G antibodies raised against CO oxidase, followed by treatment with ferritin-conjugated goat-anti-rabbit immunoglobulin G antibodies (pre-embedding labeling). Electron microscopic examination of ultrathin sections showed cytoplasmic membranes and inside-out vesicles labeled at the inner aspect, whereas the outer sides of protoplasts and membrane vesicles remained completely unlabeled. The highly sensitive protein A-gold method has been modified to allow labeling of CO oxidase with good ultrastructural preservation of the bacterial cell. Glutaraldehyde-fixed cells of P. carboxydovorans were osmificated and embedded in glycol methacrylate. Etched ultrathin sections were treated with sodium metaperiodate and incubated with the specific antibodies against CO oxidase. These antibodies were then allowed to react with protein A-gold complexes (postembedding labeling). Exponentially grown cells showed 87% of CO oxidase associated with the cytoplasmic membrane and 13% of the enzyme in the cytoplasm. The results indicate that CO oxidase is attached in vivo to the inner aspect of the cytoplasmic membrane and suggest interaction of the enzyme with a membrane-bound electron acceptor. The ratio of enzyme associated with the cytoplasmic membrane decreased to 50% in the stationary growth phase.

Aldehyde Oxidoreductases↗
PubMed1982

Action of lecithin:cholesterol acyltransferase on model lipoproteins. Preparation and characterization of model nascent high density lipoprotein.

Apolipoprotein A-I, the major protein of human plasma high density lipoprotein, is the primary activator of plasma lecithin:cholesterol acyltransferase. In vitro, the association of apolipoprotein A-I with physiological phosphatidylcholines can be catalyzed by mixing the protein and lipid with sodium cholate, which is removed by chromatography. The apolipoprotein A-I/phospholipid complex has the physical properties of an HDL, and when cholesterol is present the complex is a highly reactive substrate in the lecithin:cholesterol acyltransferase-catalyzed reaction. The relative reactivity of this complex compared with a number of other lipid-protein complexes is presented and discussed.

Apolipoprotein A-I↗