Additives and Enzyme Stability

The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers.     

Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.     

Concerning the structure of photobilirubin II.

Evidence is presented which supports the postulate that the photobilirubins IIA and IIB are diastereoisomers in which the C-3 vinyl group has cyclized intramolecularly. The evidence comes principally from proton n.m.r. spectroscopy at 400 MHz and from chemical considerations. The cyclic structures require the E-configuration at the C-4 double bond in the precursor; this is the first structural evidence for the Z leads to E isomerization in bilirubin and supports the view that the precursor (photobilirubin IA or IB) is (4E, 15Z)-bilirubin. Brief irradiation of photobilirubin II gives bilirubin, a new compound (photobilirubin III) and unchanged starting material. The various photoisomers are discussed in terms of their inter-relationships and biological fates.     

Plasma arginine vasotocin and angiotensin II in the water deprived Kelp gull (Larus dominicanus), Cape gannet (Sula capensis) and Jackass penguin (Spheniscus demersus)

1. The basal levels of the osmoregulatory hormones, arginine vasotocin (AVT) and angiotensin II (AII) were measured (by radioimmunoassay) in the plasma of conscious Kelp gulls, Cape gannets and Jackass penguins. 2. The responses of the hormones to similar degrees of hypertonicity and hypovolemia caused by water deprivation have also been determined. 3. Dehydration elevated plasma AVT and plasma AII in all three species. 4. The AVT concentration was increased by 2-4 fold and although in each case the correlation between plasma osmolality and plasma AVT was highly significant (2P less than 0.01), the sensitivity of release was greater in the gull (1.13 pg/ml per mOsm/kg) than in the gannet (0.36 pg/ml per mOsm/kg) or penguin (0.44 pg/ml per mOsm/kg). 5. Dehydration increased plasma AII 3-fold in the three bird types.     

Respiratory components in acidophilic bacteria that respire on iron

Abstract

Microorganisms capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Phylogenetically distinct organisms were shown to express spectrally distinct redox‐active biomolecules during autotrophic growth on soluble iron. A new iron‐oxidizing eubacterium, designated as strain Funis, was investigated. Strain Funis was judged to be different from other known iron‐oxidizing bacteria on the bases of comparative lipid analyses, 16S rRNA sequence analyses, and cytochrome composition studies. When grown autotrophically on ferrous ions, Funis produced conspicuous levels of a novel acid‐stable, acid‐soluble yellow cytochrome with a distinctive absorbance peak at 579 nm in the reduced state.

Stopped‐flow spectrophotometric kinetic studies were conducted on respiratory chain components isolated from cell‐free extracts of Thiobacillus ferrooxidans. Experimental results were consistent with a model where the primary oxidant of ferrous ions is a highly aggregated c‐type cytochrome that then reduces the periplasmic rusticyanin. The Fe(II)‐dependent, cytochrome c‐catalyzed reduction of the rusticyanin possessed three kinetic properties in common with corresponding intact cells that respire on iron: the same anion specificity, a similar dependence of the rate on the concentration of ferrous ions, and similar rates at saturating concentrations of ferrous ions     

Membrane lipid alterations and thermal stress in Salmonella typhimurium 7136

Salmonella typhimurium ATCC 7136 exhibited major changes in lipid composition when grown in the presence of either 0.15% sodium deoxycholate or 0.15% sodium benzoate. These lipophilic compounds had directly opposing effects on the lipid profile of the organism. The saturated/unsaturated ratio was markedly elevated in benzoate-grown cells. On the other hand, it was depressed by an even greater margin from the control after growth in the presence of deoxycholate. Adjustments in the phospholipid content of the cells were also recorded. Phosphatidylethanolamines decreased by 28 and 50% in the deoxycholate- and benzoate-grown cells, respectively. Compensatory increases in phosphatidylglycerols of 87.5 and 175% occurred, along with increases in cardiolipins of 12- and 22-fold, respectively. Deoxycholate or benzoate supplementation also altered the relative distribution of neutral lipids; again, benzoate stimulated the greater change. Compositional changes were accompanied in the organism by increased heat sensitivity, but the effect on the susceptibility of S. typhimurium to injury varied with the physical properties of the supplement used.