Class pi glutathione S-transferase from pig lung. Purification, biochemical characterization, primary structure and crystallization

A cytosolic glutathione S-transferase from pig lung was purified 210-fold to apparent homogeneity. The enzyme was classified as a class pi isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23,500, has a pI of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S-hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzyme's primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82-84% residue identity) to the rat and human class pi isoenzymes. Furthermore, there was no evidence of microheterogeneity or post-translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Singel crystals of the glutathione-S-transferase-glutathione-sulfonate complex were obtained by the hanging-drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 10.125 nm, b = 8.253 nm and c = 5.428 nm and diffract to better than 0.22 nm.     

Developmental patterns of ornithine decarboxylase activity in organogenesis phase rat embryos in culture and in utero

Summary Ornithine decarboxylase activity was measured during organogenesis in rat embryos grown in utero and whole rat conceptuses maintained in an in vitro culture system. Ornithine decarboxylase levels in vivo showed a distinct peak at embryonic age 10.5 d. Despite identical morphology, protein content, crown rump length and numbers of somites cultured embryos displayed a different developmental pattern and possessed less than half the ODC activity of that in vivo. The data suggest that the normal embryonic programming of ODC activity is significantly altered by the culture environment and that further biochemical comparisons of embryos growing in utero and in vitro may be required to evaluate properly the applicability of this technique to detailed studies of teratogenesis and developmental biology. This work was supported by NIH-5-507-RR5359-17 and a 1980 Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation.     

Genetic control of 6-phosphogluconate dehydrogenase (6-PGD) isozymes in cultivated wheat and rye

Summary The 6-phosphogluconate dehydrogenase (6-PGD) zymogram phenotypes of wheat, rye and their aneuploid derivatives were determined. Two genes involved in the production of 6-PGD isozymes were located on chromosome arms CRL (4 RL) and FRL (6 RL) of Imperial rye. On the basis of differential interactions between wheat and rye chromosomes, evidence was obtained that genes located on chromosomes 6 A, 6 BL and 7 BL control 6-PGD isozyme activities in Chinese Spring wheat. The wheat and rye 6-PGD zymogram phenotypes were indicative of homoeologous relationships between rye chromosome 6 RL to wheat chromosomes of group 6, and rye chromosome 4 RL to wheat chromosomes of group 7.     

Effects of Detergents, Proteins, and Lipids on 2'':3''-Cyclic-Nucleotide 3''-Phosphodiesterase Activity

The myelin marker 2':3'-cyclic-nucleotide 34'-phosphodiesterase (CNPase) was isolated to a lipid- and phosphate-free stage. The effects of exogenously added lipids were tested on this preparation and compared to the known stimulation of the enzyme by detergents and proteins. CNPase could be stimulated 2-3 fold by these various agents which appeared to be additive in their effect. Enzyme-protein and enzyme-lipid interactions and possible medical use of the improved assay conditions for CNPase employed in the study are discussed.     

Guidelines for safety tests on insect viruses

Guidelines for testing the safety of insect viruses for use as insecticides have been formulated in the light of the recent decade's experience and accumulation of knowledge. In the familyBaculoviridae, the nuclear polyhedrosis and granulosis viruses of Lepidoptera and sawflies (Hymenoptera) are a particularly homogeneous group which have been tested very extensively, without any evidence of hazard to man and vertebrates. It is considered, therefore, that data for viruses already tested can be used as evidence for the safety of a new virus in this group, so that only a limited series of mandatory tests are necessary on the new virus product itself. Other viruses may require the full range of tests, which include data on the identification of the virus; nature of the formulation; biological properties; manufacture; quality control; application; efficacy; residues; infectivity, toxicity and allergenicity in mammals (involving short, intermediate and long term application to various mammalian species by a variety of routes, and observation of humans who have handled the virus) and on wildlife (bees, important predators and parasites of target species, earthworms, fish and birds). It is recommended that additional non-mandatory research should be undertaken on subjects such as the behaviour of the viral genome, improved methods of identifying viruses and of testing safety.     

Internally elongated rodent α-crystallin A chain: Resulting from incomplete RNA splicing?

αA^Ins, an elongated α-crystallin A chain previously observed in rat, was present beside the normal αA chain in mouse, gerbil and hamster, which places its origin at least 30 million years ago. Like in rat the sequences of golden hamster αA^Ins and αA were found to be identical, apart from the internal insertion of 22 residues in αA^Ins. The hamster chains only differed from the rat chains by a single substitution in the inserted sequence of αA^Ins. The origin of αA^Ins, by insertion of 22 residues in an otherwise unchanged αA chain, and its rigid evolutionary conservation are most easily explained by assuming the incomplete removal of a putative intervening sequence from the precursor mRNA of αA, leaving an intracistronic insert of 66 nucleotides in part of the eventually translated mRNA.     

Isolation of the globular region of the subcomponent q of the C1 component of complement.

Digestion after heat treatment of the subcomponent q of the C1 component of complement by collagenase leads to the isolation of the globular region of the protein. This product ('heads') is composed of three chains giving an overall molecular weight of about 57000. About half of the collagen-like region present in C1 q is lost after digestion. The 'heads' are shown to be soluble and hemolytically active products.     

The action of beta-galactosidase (Escherichia coli) on allolactose.

The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.