Applicability of the induced-fit model to glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. Study of the binding of oxidized nicotinamide adenine dinucleotide and nicotinamide 8-bromoadenine dinucleotide

A new method of calculation, based on a direct fitting of the protein fluorescence intensity observed upon coenzyme binding (H.-P. Lutz, unpublished results), is used to study the negative cooperative behavior of glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. The calculation procedure simultaneously elaborates data obtained for four different protein concentrations, and it is able to compare different models by computing the minimal and critical sum of squares. Using this approach, it is shown that the induced-fit model [Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5,365] and the dimer of dimer model [Malhotra, O. P., & Bernhard, S. A. (1968) J. Biol. Chem. 243, 1243-1252] can both be applied for explaining the negative cooperativity observed upon coenzyme binding to sturgeon glyceraldehyde-3-phosphate dehydrogenase. In addition to the progressive modification of the binding affinity during ligand binding, different maximal fluorescence quenchings for the binding steps must be postulated; and furthermore, the binding capability decreases by decreasing the protein concentration. The fact that the induced-fit model can also be applied is rather in contradiction with the view generally accepted of a dimer of dimer structure of sturgeon glyceraldehyde-3-phosphate dehydrogenase. By use of the same approach, nicotinamide 8-bromoadenine dinucleotide is shown to bind to glyceraldehyde-3-phosphate dehydrogenase from sturgeon in a negative cooperative manner.     

Comparison of the effect of temperature on kidney cortex mitochondria from rabbit,dog, pig,and human: Arrhenius plots of ADP-stimulated respiration

The effect of temperature on the rate of ADP-stimulated respiration of mitochondria from dog, rabbit, pig, and human kidney cortex mitochondria was plotted according to the Arrhenius relationship. The temperature at which the plot demonstrated a break was at 15 °C for mitochondria from dog, pig, and human kidneys. The discontinuity occurred at 10 °C or less for mitochondria from rabbit kidneys. This difference suggests that mitochondria from rabbit kidneys undergo a lipid-phase transition at lower temperatures than for other species commonly used in experimental renal preservation. The implications of this difference suggest caution in using results obtained with rabbit kidneys for comparison to results obtained from hypothermic renal preservation of other species kidneys. Apparent fluidization of dog kidney mitochondrial membranes with adamantine abolished the discontinuity in the Arrhenius plot.     

Hg ++ - a DCMU independent electron acceptor of photosystem II

Mercuric chloride functions as a direct electron acceptor from the quencher of fluorescence in Photosystem II. The photoreduction of ferricyanide, dichlorophenol-indophenol or methyl viologen is inhibited by mercuric ion while oxygen evolution is uneffected. Mercuric chloride supported oxygen evolution (mercury Hill reaction) is not prevented by DCMU or other similar electron transport inhibitors.     

Über die Herkunft „synaptischer“ Bläschen in neurosekretorischen Axonen

Zusammenfassung In der Neurohypophyse fetaler und neugeborener Ratten entsteht die Mehrzahl der synaptischen Bläschen aus Erweiterungen der Neurotubuli. Ferner können pinocytotische Bläschen als synaptische Vesikel imponieren. Die Bläschenbildung aus Membranen von Elementargranula (vgl. Herlant, 1967) tritt dagegen in den Hintergrund. Ein Auftreten von Vesikeln im Innern von Elementargranula wurde nicht beobachtet.

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The origin of synaptic vesicles in neurosecretory axons

Summary In the neurohypophysis of fetal and newborn rats the majority of synaptic vesicles originate from dilatations of neurotubuli. Moreover, pinocytotic invaginations give rise to synaptic vesicles. Evaginations of elementary granule membranes, as described by Herlant (1967), are seldom to be found and do not seem to play an important role in the formation of synaptic vesicles. The occurrence of vesicles within elementary granules was not observed.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Induction of Lambda-Bacteriophage in Escherichia coli as a Screening Test for Potential Antitumor Agents

A simple, rapid, quantitative test procedure to measure induction of phage production in lysogenic Escherichia coli K-12 (λ) was described. This test was used in a study of 209 substances, including antibiotics, pyrimirines, purines, alkylating agents, thiols, amino acids, vitamins, and miscellaneous compounds. Minimal inducing concentrations for the 26 (12.5% of total tested) substances found to be effective inducing agents, as well as a listing of the inactive compounds, are presented. Since 21 of the 26 active agents reportedly have antineoplastic activity in rodent tumor systems, it was concluded that the induction test may provide a useful screen for the detection of potentially useful antitumor compounds.     

The Ubiquitin System in Higher and Lower Plants — Pathways in Protein Metabolism

The multiple biological functions of the small polypeptide ubiquitin are mirrored by its unparalleled conservation on the amino acid and gene organization level. During the last years, it has become widely accepted that ubiquitin is an essential component in the ATP-dependent nonlysosomal protein degradation pathway occurring in all eukaryotic organisms. As turnover, consisting of protein synthesis and disassembly, is a central and vital process for each living cell, ubiquitin-mediated proteolysis is of enormous physiological value. The components of the ubiquitin ligation system have been characterized skillfully in plant and animal cells, but at the moment many questions remain as to how the high degree of specificity that is necessary for the regulation of intracellular breakdown is ensured. The recent hypotheses and models proposed for the basic mechanisms of protein recognition, conjugation and degradation will be discussed in detail. The existence of ubiquitin-protein conjugates which are not rapidly degraded clearly suggested that the role of ubiquitin is not restricted in its implication for protein turnover. Alterations of DNA structure, specific cell recognition mechanisms and cytoskeletal variations were observed as further ubiquitin-dependent processes which are not directly coupled to protein degradation.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Synthesis and biological activity of a biotinylated vasopressin analog

To study vasopressin receptor-mediated endocytosis using electronmicroscopy methods and to develop avidin affinity columns for receptor purification, we synthesized and tested the biological properties of a biotinylated vasopressin (VP) analog [1-(2-mercapto) propionic acid] 8-[lysine-N6-biotin] VP (B-MLVP). B-MLVP was prepared by coupling biotin to the epsilon amine of the lysine residue in [1-(2-mercapto) propionic acid] 8-(lysine) VP (MLVP). The structure of HPLC purified B-MLVP was confirmed by fast atom bombardment mass spectrometry. B-MLVP effectively competed for arginine vasopressin (AVP) binding sites in canine renal plasma membranes on the surface of LLC-PK1 kidney cells. Dissociation constants of 15 nM and 202 nM were calculated from the results of competition binding assays conducted with membranes and cells, respectively. B-MLVP stimulated adenylate cyclase activity and elevated cellular 3',5',cyclic-AMP (cAMP) content in a manner similar to AVP, indicating it is an agonist of VP action in renal tissue. These observations indicate that B-MLVP is an agonist of VP action and may be used to study renal VP receptors by employing avidin coupled to various reporter groups.