Supercoil-stabilized left-handed DNA in the plasmid (dA-dT)16 insert formed in the presence of Ni2+

The (dA-dT)16 insert of the plasmid pAT32 was probed with diethyl pyrocarbonate (DEPC) and nuclease Bal3l in the presence of Ni2+ known to be able to induce transition to left-handed conformation in the synthetic poly(dA-dT).poly(dA-T). It has been shown that this insert in a supercoiled plasmid displays a DEPC modification pattern characteristic of left-handed DNA under conditions not sufficient to induce a left-handed structure in the linear plasmid and poly(dA-dT).poly(dA-T).     

Utilization of alanine for glucose formation in isolated rabbit kidney-cortex tubules

In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as glucose precursor, when added as a sole substrate. However, this amino acid decreases gluconeogenesis from low (up to 1 mM) 2-oxoglutarate concentrations and stimulates this process at higher (2.5-10 mM) ketoacid contents in the suspension medium. Aminooxyacetate, an inhibitor of aminotransferases, abolishes both inhibitory and stimulatory effects of L-alanine on glucose formation. The addition of 2-oxoglutarate increases the incorporation of L-[U-14C]alanine to glucose from 8- to 123-fold, depending upon the ketoacid and alanine concentrations used. In contrast, nonlabelled L-alanine decreases the incorporation of low [U-14C)2-oxoglutarate concentrations into glucose, while it does not affect contribution of 5 mM ketoacid to gluconeogenesis. The data indicate that (i) in the presence of 2-oxoglutarate L-alanine is utilized as glucose precursor in rabbit renal tubules and (ii) this amino acid may decrease the contribution of low extracellular concentrations of the ketoacid to gluconeogenesis.     

Isolation of a cDNA encoding the human GM2 activator protein

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.     

Y1 and Y2 receptors for neuropeptide Y

By using monoiodinated radioligands of both intact neuropeptide Y (NPY) and of a long C-terminal fragment, NPY13-36, two subtypes of binding sites, which differ in affinity and specificity, have been characterized. The Y1 type of binding site, characterized on a human neuroblastoma cell line, MC-IXC, and a rat pheochromocytoma cell line, PC-12, binds NPY with a dissociation constant (Kd) of a few nanomolar but does not bind NPY13-36. The Y2 type of binding site, characterized on porcine hippocampal membranes and on another human neuroblastoma cell line, SMS-MSN, is of higher affinity and binds both NPY and NPY13-36. None of the binding sites distinguish between NPY and the homologous peptide YY (PYY). It is concluded that NPY/PYY-binding sites occur in two subtypes which may represent two types of physiological receptors.     

Interaction of rat alpha-fetoprotein and albumin with polyunsaturated and other fatty acids: determination of apparent association constants

The interaction of fatty acids with rat alpha-fetoprotein and albumin was measured using a partition equilibrium method. alpha-Fetoprotein (AFP) displays one high-affinity binding site for fatty acids and albumin near two binding sites. The AFP association constants for most fatty acids were similar to those of albumin (in the 10(7) M-1 range) whereas for docosahexaenoic acid it was 9.7 x 10(8) M-1, about 50-fold higher than that corresponding to albumin. This difference justifies docosahexaenoic acid in fetal or neonatal serum being mainly bound to AFP and can indicate a highly specific role of AFP in the transport of this fatty acid.     

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.     

Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli

Spheroplasts from aerobically grown wild-type Paracoccus denitrificans cells respire with succinate despite specific inhibition of the cytochrome bc1 complex by myxothiazol. Coupled to this activity, which involves only b-type cytochromes, there is translocation of 1.5-1.9 h+/e- across the cytoplasmic membrane. Similar H+ translocation ratios are observed during oxidation of ubiquinol in spheroplasts from aerobically grown mutants of Paracoccus lacking cytochrome c oxidase, or deficient in cytochrome c, as well as in a strain of E. coli from which cytochrome d was deleted. These observations show that the cytochrome o complex is a proton pump much like cytochrome aa3 to which it is structurally related.     

Protein S binding in relation to the subunit composition of human C4b-binding protein

The human regulatory complement component C4b-binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K-dependent protein S. The major form of C4BP is composed of 7 identical, disulfide-linked 70 kDa subunits (alpha-chains), the arrangement of which gives the C4BP molecule a spider-like appearance. Recently, we identified a unique 45 kDa subunit (beta-chain) in C4BP. We have now isolated a subpopulation of C4BP, which does not bind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the beta-chain. Another lower molecular weight form of C4BP was also purified. It contained the beta-chain and was efficient in binding protein S. Its subunit composition was judged to comprise six alpha-chains and one beta-chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis-a-vis subunit composition and/or protein S binding ability and provide support for the concept that the beta-chain of C4BP contains the single protein S binding site.     

A cDNA clone encoding the precursor for a 10.2 kDa photosystem I polypeptide of barley

Two cDNA clones for the barley photosystem I polypeptide which migrates with an apparent molecular mass of 9.5 kDa on SDS-polyacrylamide gels have been isolated using antibodies and an oligonucleotide probe. The determined N-terminal amino acid sequence for the mature polypeptide confirms the identification of the clones. The 644 base-pair sequence of one of the clones contains one large open reading frame coding for a 14 882 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 193 Da. The hydropathy plot of the polypeptide shows one membrane-spanning region with a predicted -helix secondary structure. The gene for the 9.5 kDa polypeptide has been designated PsaH.