Modification in the purification of the sex steroid-binding protein of human serum by affinity chromatography

A purification procedure for the sex steroid-binding protein of human serum is described. The procedure is significantly superior to that recently published (K. E. Mickelson, D. C. Teller, and P. H. Pétra, 1978, Biochemistry17, 1409–1415) and should replace it for the routine preparation of homogeneous protein in relatively larger quantities. The steps involved diethylaminoethyl-cellulose chromatography, affinity chromatography on 5α-dihydrotestosterone-17α-hexanyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The most important difference between this new procedure and that previously published is the affinity adsorbent with contains the steroid covalently linked at the 17α-position rather than the 17β-position. This modification allows the purification of at least 12 mg of homogeneous protein per preparation with a 63% total yield. The properties of the homogeneous protein are the same as previously described.     

Stimulation of the initiation of DNA synthesis and cell division in several cultured mouse cell types : Effect of growth-promoting hormones and nutrients

The ability of prostaglandin F_2α (PGF_2α) and other prostaglandins to stimulate the initiation of DNA synthesis in quiescent cultures of various mouse fibroblastic cell types has been investigated. PGF_2α was found to be more effective than the other prostaglandins. Most cell types, with the exception of BALB/c 3T3, responded to PGF_2α. Addition of PGF_2α in combination with insulin resulted in a synergistic increase in the proportion of cells synthesizing DNA. The effect of nutrients on the stimulation of the initiation of DNA synthesis has been examined in detail; it was found that Swiss 3T3 cells showed a requirement for hypoxanthine and vitamin B₁₂ whereas Swiss 3T6 cells demonstrated a stringent requirement for vitamin B₁₂ only. The effect of prostaglandin precursors, synthetic analogues of the prostaglandin endoperoxides and inhibitors of prostaglandin synthesis was also examined in two cell types. The effect of PGF_2α was compared with that of two polypeptide growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF) in Swiss 3T6 cells grown in 0.0025% (v/v) serum. In combination with insulin each of these three growth factors stimulated the initiation of DNA synthesis in approximately the same number of cells.     

Light Effects in Yeast: Evidence for Participation of Cytochromes in Photoinhibition of Growth and Transport in Saccharomyces cerevisiae Cultured at Low Temperatures

Visible light of moderate intensity inhibits growth, respiration, protein synthesis, and membrane transport in bakers' yeast and has a deleterious effect on membrane integrity. The results of this study indicate that these effects require the presence of cytochromes b and a/a(3). The light sensitivities of growth rate and [(14)C]histidine uptake in wild-type rho(+) Y185 and D225-5A strains of Saccharomyces cerevisiae were compared with those in a variety of mutants lacking cytochrome b or a/a(3) or both; a close correlation was found between the presence of these respiratory pigments and photosensitivity. Thus, strain TL5-3C, a nuclear petite lacking cytochromes b, a, and a(3), was resistant to light; strain GL5-6A, another nuclear petite having reduced amounts of cytochromes a and a(3), was partially resistant; strains MB127-20C and MB1-6C, nuclear petites lacking only cytochrome b, were also only partially resistant to light; whereas mutants containing all three cytochromes but having their respiratory chain either nonfunctional (strain ZK3-6B) or uncoupled (strain 18-27/t12) were fully sensitive to light. Finally, an equal-energy, broad-band action spectrum for the light inhibition of growth and transport indicated that blue light (408 nm) was most effective; these wavelengths correspond to the Soret region of the cytochrome absorption spectrum. The results suggest, therefore, that the yeast cytochromes b, a, and a(3) are the primary photoreceptors for the inhibitory effects of light and, perhaps, for other processes, such as the entrainment of biological rhythms in this species.     

Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. Involvement of non-carbon monoxide-forming mechanisms

Degradation of intrinsic hepatic [(14)C]haem was analysed as (14)CO formation in living rats and in hepatic microsomal fractions prepared from these animals 16h after pulse-labelling with 5-amino[5-(14)C]laevulinic acid, a precursor that labels bridge carbons of haem in non-erythroid tissues. NADPH-catalysed peroxidation of microsomal lipids in vitro (measured as malondialdehyde) was accompanied by loss of cytochrome P-450 and microsome-associated [(14)C]haem (largely cytochrome P-450 haem), but little (14)CO formation. No additional (14)CO was formed when carbon tetrachloride and 2-allyl-2-isopropylacetamide were added to stimulate lipid peroxidation and increase loss of cytochrome P-450 [(14)C]haem. Because the latter effect persisted despite inhibition of lipid peroxidation with MnCl(2) or phenyl-t-butylnitrone(a spin-trapping agent for free radicals), it was concluded that carbon tetrachloride, as reported for 2-allyl-2-isopropylacetamide, may promote loss of cytochrome P-450 haem through a non-CO-forming mechanism independent of lipid peroxidation. By comparison with breakdown of intrinsic haem, catabolism of [(14)C]methaemalbumin by microsomal haem oxygenase in vitro produced equimolar quantities of (14)CO and bilirubin, although these catabolites reflected only 18% of the degraded [(14)C]haem. This value was increased to 100% by addition of MnCl(2), which suggests that lipid peroxidation may be involved in degradation of exogenous haem to products other than CO. Phenyl-t-butylnitrone completely blocked haem oxygenase activity, which suggests that hydroxy free radicals may represent a species of active oxygen used by this enzyme system. After administration of carbon tetrachloride or 2-allyl-2-isopropylacetamide to labelled rats, hepatic [(14)C]haem was decreased and haem oxygenase activity was unchanged; however, (14)CO excretion was either unchanged (carbon tetrachloride) or decreased (2-allyl-2-isopropylacetamide). These changes were unaffected by cycloheximide pretreatment. From the lack of parallel losses of cytochrome P-450 [(14)C]haem and (14)CO excretion, one may infer that an important fraction of hepatic [(14)C]haem in normal rats is degraded by endogenous pathways not involving CO. We conclude that carbon tetrachloride and 2-allyl-2-isopropylacetamide accelerate catabolism of cytochrome P-450 haem through mechanisms that do not yield CO as an end product, and that are insensitive to cycloheximide and independent of haem oxygenase activity.     

Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology

Summary This is the third in a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxribonucleate)-3-nucleotidohydrolase, EC 3.1.4.4 (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article describes the structure of the nuclease and of a nuclease-inhibitor complex as determined by x-ray crystallography. The crystal structures are correlated with some of the known chemical and enzymological properties of the enzyme, and the three areas combined to propose a mechanism of action.This article is the third in a series of four devoted to the stapholoccal nuclease. Reviews concerning its isolation and enzymology (1) as well as the features of its ligand binding site (2) have appeared in previous issues. Work from this laboratory has been supported by grants from the National Institute of General Medical Sciences, NIH and from the Robert A. Welch Foundation to F. A. Cotton and E. E. Hazen, Jr.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterations were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.     

High capacity gel preparative electrophoresis for purification of fragments of genomic DNA

We have designed a high-capacity gel electrophoretic device for the purification of large amounts of restriction endonuclease fragments of genomic DNA. This device exploits the high resolution of gel electrophoresis in conjunction with an electronic system permitting discontinuous sample elution over a large gel surface area. This feature preserves resolution and greatly increases capacity and yield. The resulting DNA fractions may be used in restriction endonuclease, ligation, transfection, and transformation reactions without further extensive manipulation. Furthermore, DNA fragments of a broad size range are recovered with high efficiency from the gel.