Affinity chromatography of the phosphatidylcholine exchange protein from bovine liver

Affinity chromatography has been used to purify the phosphatidylcholine exchange protein from bovine liver. The affinity resin consisted of 1-acyl-2-(9-carboxy)nonyl-glycero-3-phosphocholine linked to AH-Sepharose 4 B via the carboxyl group. Application of a crude exchange protein fraction to the affinity column resulted in a complete adsorption of the phosphatidylcholine exchange protein. The exchange protein eluted with a buffer containing 0.15% sodium deoxycholate. The most active fraction was 130-fold purified and accounted for 62% of the activity.     

Kinetic properties of pyruvate kinase isolated from rat hepatic tumours.

The results demonstrate the existence of L and M forms of pyruvate kinase in rat hepatomas. Tumours were induced by feeding N-Nitrosodiethylamine. The kinetic properties of the L-type tumour enzyme was markedly different from the L-enzyme form found in normal liver. The L-form of tumour enzyme was purified by DEAE cellulose-Sephadex G200 chromatography (Sp. activity 41 units/mg). $\text{MgADP}{}^{\mathrm{?}}\text{ADP}{}^{\mathrm{2?}}$ of $\text{20}\text{1}$ gave optimum activity for both the intrinsic and F1,6di-P stimulated reactions. ATP did not inhibit the enzyme. Alanine (2.5 nM) caused 60% inhibition at low PEP concentrations (0.25 mM). The homotropic effector (PEP) exhibited a complex allosteric pattern and saturation kinetics were not observed for either the intrinsic or F1,6di-P stimulated reactions with PEP concentrations as high as 10 mM.     

Neurotensin is produced by and secreted from classic small cell lung cancer cells

The presence of neurotensin in various human tumor cell lines was investigated by radioimmunoassay. High concentrations (0.06-5.1 pmol/mg protein) were detected in 50% of the classic but not variant small cell lung cancer or other human tumor cell lines examined. Biochemical studies indicated that the main peak of immunoreactivity coeluted with synthetic neurotensin using gel filtration and high pressure liquid chromatography techniques. Also, the rate of neurotensin secretion increased approximately 2-fold when theophylline was added which elevated intracellular levels of cAMP 4-fold. Because neurotensin is present in and secreted from many classic small cell lung cancer cells, it may function as a regulatory peptide in this disease.     

A rapid procedure to detect in situ DNA/RNA hybrids

We developed a new method for detecting DNA/RNA hybrids formed $\text{in}\text{situ}$ using $\text{anti-}\text{DNA}\text{RNA}$ antibodies and the Peroxidase-antiperoxidase immunohistochemical procedure. Using RNA synthesised $\text{in}\text{vitro}$ from cloned $\text{Drosophila}$ histone genes (pDm 500H), we localized by this procedure, the histone genes to the 39 D-E region of the left arm of the second chromosome. This method has several advantages compared to conventional procedures.     

Diguanosine nucleotides of fungi that regulate RNA polymerases isolated and partially characterised.

Three unusual phosphorylated diguanosine compounds called ‘hot spots’ HS-1, HS-2 and HS-3 (ref. LéJohn, H.B. Proc. Can. Fed. Biol. Soc. 18, 159, 1975) have been isolated as acid-soluble materials from several fungi, $\text{Achlya, Blastocladiella emersonii, Aspergillus niger}$ and $\text{Rhizopus stolonifer}$ in their vegetative phase. The nucleotides were purified from acid extracts of $\text{Achlya}$ and $\text{Blastocladiella}$. The tentative structures of HS-3 and HS-2 determined are GppppG and GppppGp. HS-1 structure is still in doubt but it is related to HS-2. The structures were deduced from enzymatic digestion and UV analyses of the products, molar ratios of guanosine and phosphate, and chromatographic behaviour on PEI-cellulose. All three compounds accumulated in an inverse manner with rates of RNA synthesis and directly with rates of protein synthesis. The acid-soluble pools of the three compounds fluctuated during the life cycle of $\text{Achlya}$, and just prior to sporulation, were excreted into the medium. HS-2 was convertible to HS-3 by acid hydrolysis.     

The oxidation of tricarboxylic acid cycle intermediates, with particular reference to isocitrate, by intact mitochondria isolated from the liver of the American eel, Anguilla rostrata leSueur.

The oxidation of exogenously added substrates has been studied in intact liver mitochondria isolated from the American eel, Anguilla rostrata. These data, coupled to determinations of the activity and localization of critical tricarboxylic acid (TCA) cycle enzymes, have been used to propose a pathway for the eel liver TCA cycle. (1) Isocitric, α-ketoglutaric, succinic, and malic acids are oxidized at essentially equivalent rates by eel mitochondria, with normal ADP:O and respiratory control ratios. No oxidation of citric, oxaloacetic, or pyruvic acids was detected when added alone or with malate, although oxaloacetic acid + pyruvic acid was oxidized but at a much reduced rate. (2) Radioactively labeled isocitrate was incorporated into at least α-ketoglutaric, succinic, and malic acids, indicating the eel liver TCA cycle is normal between isocitrate and malate. (3) No activity of the NAD-linked isocitrate dehydrogenase (IDH) could be detected, but NADP-IDH activities were higher in the mitochondria than cytosolic fractions. An active NADPH:NAD transhydrogenase was localized to the mitochondrial compartment. (4) These data suggest an important role for the NADP-IDH:transhydrogenase enzyme couple in eel liver TCA cycle function, and a pathway incorporating these ideas is proposed.