Kinetics of molecular chaperone action.

Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by relaxation times of 27 seconds and 200 seconds with 2 microM DnaK and 0.

Evolutionary relationships among pyridoxal-5'-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families.

Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis.

Homology of 1-aminocyclopropane-1-carboxylate synthase, 8-amino-7-oxononanoate synthase, 2-amino-6-caprolactam racemase, 2,2-dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and isopenicillin-N-epimerase with aminotransferases.

Profile analysis showed the title enzymes to be homologous with the aminotransferases. 1-Aminocyclopropane-1-carboxylate synthase is closely related to subgroup I of aminotransferases which includes aspartate, alanine, histidinol-phosphate, tyrosine and phenylalanine aminotransferase. 2,2-Dialkylglycine decarboxylase, glutamate-1-semialdehyde 2,1-aminomutase and 2-amino-6-caprolactam racemase are most similar to subgroup II which comprises aminotransferases with omega-amino acids as substrates.

The mature form of imported mitochondrial proteins undergoes conformational changes upon binding to isolated mitochondria.

Mature mitochondrial proteins (aspartate aminotransferase, malate dehydrogenase, hydroxyacyl coenzyme A dehydrogenase, creatine kinase) and cytosolic proteins (aldolase, glyceraldehyde-3-phosphate dehydrogenase) with a basic pI were found to bind to isolated mitochondria, electrostatic interactions being mainly responsible for their binding. Mitochondrial aspartate aminotransferase bound with a Kd' of 30 nM in 0.6 M sorbitol, 20 mM Hepes/KOH, pH 7.

Reaction of imidazole-citrate-deformed glycogen phosphorylase with amino acids.

Incubation of phosphorylase with L-valine in the presence of 0.4 M imidazole citrate results in a time-dependent decrease in the absorption of the enzyme-bound cofactor pyridoxal 5'-phosphate at 333 nm and the generation of a new absorption maximum at 415 nm which appears to be due to an enzyme-bound coenzyme-amino-acid aldimine adduct. Consequently, the enzyme is inactivated to less than 10% of its initial activity.

Aminotransferases: demonstration of homology and division into evolutionary subgroups.

A total of 150 amino acid sequences of vitamin B6-dependent enzymes are known to date, the largest contingent being furnished by the aminotransferases with 51 sequences of 14 different enzymes. All aminotransferase sequences were aligned by using algorithms for sequence comparison, hydropathy patterns and secondary structure predictions. The aminotransferases could be divided into four subgroups on the basis of their mutual structural relatedness.

Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site.

Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxidants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldolase from rabbit muscle the polypeptide chain had been found to be crosslinked at active-site Lys229 (Schiff base forming with substrate) and Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G.

Mutant aspartate aminotransferase (K258H) without pyridoxal-5'-phosphate-binding lysine residue. Structural and catalytic properties.

If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H.

Homology of pyridoxal-5'-phosphate-dependent aminotransferases with the cobC (cobalamin synthesis), nifS (nitrogen fixation), pabC (p-aminobenzoate synthesis) and malY (abolishing endogenous induction of the maltose system) gene products.

Bacterial deletion mutants have indicated that the gene products of cobC, nifS, pabC and malY participate in important metabolic pathways, i.e. cobalamin synthesis, nitrogen fixation, synthesis of p-aminobenzoate and the regulation of the maltose system, respectively.

Growth of Candida utilis in solid state fermentation.

In this work, the growth of the yeast Candida utilis on different solid substrate (wheat bran) and supports (sugarcane bagasse and Amberlite resin) imbibed with a liquid culture medium was studied. Growth was followed by sugars consumption, carbon dioxide production rate (CDPR) and cell count. The results showed the ability of the yeast to grow on the three solid media with fairly good viability and total dextrose consumption in the case of sugarcane bagasse and Amberlite, and partial consumption of wheat bran sugars.

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4.

Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium.

Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat-shock protein DnaK.

On expression of the cDNA encoding the precursor of chicken mitochondrial aspartate aminotransferase (pmAspAT) in Escherichia coli, the bulk of pmAspAT was found to be associated with the 70-kDa heat-shock protein DnaK which is closely related to mitochondrial 70-kDa heat-shock protein (HSP70). Purification protocols for the DnaK/pmAspAT complex and its individual components were elaborated. The complex dissociated on treatment with MgATP or at pH 5.

High-yield production of valepotriates by hairy root cultures of Valeriana officnalis L. var. sambucifolia Mikan.

Hairy root cultures of Valeriana officinalis var. sambucifolia were established by infection of sterile plantlets with Agrobacterium rhizogenes strain R1601 The transformed roots were grown in 10 different, hormone-free liquid media and the isovaltrate, valtrate, didrovaltrate, isovaleroxyhydroxydidrovaltrate content was quantified by HPLC. Valepotriates were entirely retained inside the root tissues.

Mechanism of racemization of amino acids by aspartate aminotransferase.

Aspartate aminotransferase (mitochondrial isoenzyme from chicken) has been found to racemize very slowly dicarboxylic amino acid substrates in the presence of their cognate oxo acids [Kochhar, S. & Christen, P. (1988) Eur.

The open/closed conformational equilibrium of aspartate aminotransferase. Studies in the crystalline state and with a fluorescent probe in solution.

Aspartate aminotransferase undergoes major shifts in the conformational equilibrium of the protein matrix during transamination. The present study defines the two conformational states of the enzyme by crystallographic analysis, examines the conditions under which the enzyme crystallizes in each of these conformations, and correlates these conditions with the conformational behaviour of the enzyme in solution, as monitored by a fluorescent reporter group. Cocrystallization of chicken mitochondrial aspartate aminotransferase with inhibitors and covalent coenzymesubstrate adducts yields three different crystal forms.

The precursor of mitochondrial aspartate aminotransferase is imported into mitochondria faster than the homologous cytosolic isoenzyme with the same presequence attached.

Mitochondrial and cytosolic aspartate aminotransferase (AspAT) are homologous proteins with identically folded polypeptide chains. The cDNAs of the two isoenzymes of chicken were used to express the following proteins in yeast: the precursor of mitochondrial AspAT, mature mitochondrial AspAT, and two chimeric proteins in one of which (pc) the presequence of the precursor was attached to the entire cytosolic isoenzyme and in the other one (pmc) the N-terminal segment (amino acid residues -22 to 23) of the precursor was linked to the slightly truncated cytosolic isoenzyme (residues 34 to 412). All presequence containing proteins were imported into the mitochondria and processed to the mature form whereas mature mitochondrial AspAT remained in the cytosol.

Stability of prednisolone and prednisolone acetate in various vehicles used in semi-solid topical preparations.

The stability of prednisolone and prednisolone acetate has been investigated at 37 degrees C in seven semi-solid vehicles, including almond oil, beeswax, Cremophor RH 40, Lanette N, and hydrogel formulations of hydroxy-propylcellulose, carbomer and bentonite. Extraction methods and a high-performance liquid chromatographic (HPLC) procedure have been developed in order to assay the content of undegraded steroid at time 0 and after 28 days. Prednisolone acetate showed superior stability compared to prednisolone.

Structure of the genes of two homologous intracellularly heterotopic isoenzymes. Cytosolic and mitochondrial aspartate aminotransferase of chicken.

The genes of mitochondrial and cytosolic aspartate aminotransferase of chicken were cloned and sequenced. In both genes nine exons encode the mature enzyme. The additional exon for the N-terminal presequence that directs mitochondrial aspartate aminotransferase into the mitochondria is separated by the largest intron from the rest of the gene.

Alkaloids of hairy root cultures of a Datura candida hybrid.

From the in vitro hairy root cultures of a Datura candida hybrid, 19 tropane alkaloids have been identified using capillary gas-liquid chromatography and mass spectrometry. As in the parent plants, scopolamine is the major alkaloid. Two hitherto undescribed alkaloids have been detected and their structure tentatively characterised on the basis of their mass spectral fragmentations.

Ethanol extraction by supported liquid membrane during fermentation.

A supported liquid membrane system was developed for the extraction of ethanol during semicontinuous fermentation of Saccharomyces bayanus. it consisted of a porous Teflon sheet as support, soaked with isotridecanol. This assembly permitted combining biocompatibility, permeation efficiency, and stability.

Influence of monoclonal immunoglobulins in three different methods for inorganic phosphorus.

Three methods for the determination of inorganic phosphorus were compared in order to evaluate the frequency of falsely high concentrations of inorganic phosphorus in 102 sera containing monoclonal immunoglobulins. The results for the three methods using neat sera were compared with those obtained from protein-free filtrates of the same sera. For the neat sera erroneously high results were encountered with the molybdate method in about 10% (11/102) of the cases.

Aspartate aminotransferase with the pyridoxal-5'-phosphate-binding lysine residue replaced by histidine retains partial catalytic competence.

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site-directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2-oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (lambda max = 330 nm) to that of the pyridoxal form (lambda max = 330/392 nm).

[Atypical sciatica].

A 46-year old patient had suffered from progressive nocturnal sciatica and motor weakness of the left lower extremity for three years. The physical examination revealed a discrete neurologic deficit of the left L5 and S1 root. By CT a small presacral mass between the L5 and S1 roots was found.