Radiation inactivation of ribonucleotide reductase, an enzyme with a stable free radical.

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of approximately 210 kDa and the other of approximately 20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.

Improved purification protocol of the HSV-1 protease catalytic domain, using immunoaffinity.

The catalytic domain of herpes simplex virus protease was expressed in baculovirus-infected cells and purified in milligram quantities by ion-exchange and size-exclusion chromatography. The usefulness of this material was limited by the presence of a contaminating proteolytic activity, which caused time-dependent degradation of the protease. As a result we decided to explore an alternative approach to purification. Specific monoclonal antibodies were produced and evaluated by surface plasmon resonance as ligands for immunoaffinity chromatography. One monoclonal antibody, 6H4, was chosen for coupling to an affinity support, and the resulting column allowed us to obtain a pure and stable enzyme. Immunoaffinity chromatography of herpes simplex virus type 1 protease resulted in successful elimination of the contaminating protease activity. Moreover the immunoaffinity column permitted the isolation of stable and pure enzyme in a one-column procedure.

A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo.

Herpes simplex viruses (HSV) types 1 and 2 encode their own ribonucleotide reductases (RNRs) (EC 1.17.4.1) to convert ribonucleoside diphosphates into the corresponding deoxyribonucleotides. Like other iron-dependent RNRs, the viral enzyme is formed by the reversible association of two distinct homodimeric subunits. The carboxy terminus of the RNR small subunit (R2) is critical for subunit association and synthetic peptides containing these amino-acid sequences selectively inhibit the viral enzyme by preventing subunit association. Increasing evidence indicates that the HSV RNR is important for virulence and reactivation from latency. Previously, we reported on the design of HSV RNR inhibitors with enhanced inhibitory potency in vitro. We now report on BILD 1263, which to our knowledge is the first HSV RNR subunit-association inhibitor with antiviral activity in vivo. This compound suppresses the replication of HSV-1, HSV-2 and acyclovir-resistant HSV strains in cell culture, and also strongly potentiates the antiviral activity of acyclovir. Most importantly, its anti-herpetic activity is shown in a murine ocular model of HSV-1-induced keratitis, providing an example of potent nonsubstrate-based antiviral agents that prevent protein-protein interactions. The unique antiviral properties of BILD 1263 may lead to the design of new strategies to treat herpesvirus infections in humans.

A solid-phase assay for the binding of peptidic subunit association inhibitors to the herpes simplex virus ribonucleotide reductase large subunit.

The herpes simplex virus (HSV) ribonucleotide reductase is comprised of two nonidentical homodimeric subunits whose association is essential for enzymatic activity. In order to evaluate the affinity of a series of peptidic inhibitors with the enzyme's large subunit (R1), we have developed a sensitive solid-phase binding assay. The assay entails the use of a nonneutralizing antibody directed against the R1 subunit of the enzyme to immobilize either the native holoenzyme from HSV-1-infected cells or a recombinantly expressed HSV-2 R1 subunit. In either case, the radioiodinated peptidic inhibitor 125I-desamino-Tyr-(N-methyl)-Val-Ile-Asp-(gamma-N,N-diethyl)-Asp-Leu demonstrated specific, saturable binding to the HSV R1 that could be competed by the nonapeptide Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu corresponding to the C-terminal sequence of the HSV ribonucleotide reductase small subunit (R2) or by recombinant HSV R2, but not by C-terminally truncated HSV R2 or murine R2. Our results provide direct evidence that inhibitors based on the carboxy-terminal amino acid sequence of HSV R2 compete with intact HSV R2 for a common binding site on HSV R1. The utility and sensitivity of this binding assay were further demonstrated by the ability to detect and discriminate ribonucleotide reductase inhibitors in the low nanomolar range.

Interactions between endothelin and atrial natriuretic factor in the rat aortic ring preparation.

The potential interaction (s) between atrial natriuretic factor (ANF) and porcine--human endothelin (ET-1) was investigated in the endothelium-denuded rat aortic ring preparation. ET-1 produced a sustained contraction of aortic rings with an ED50 of 3.6 +/- 0.49 x 10(-9) M. Within the concentration range of 10(-9) to 10(-7) M, both rat ANF 103-126 and rat ANF 99-126 when preincubated with tissues reduced the contractile efficacy of ET-1 especially at low concentrations resulting in a small but significant rightward shift of the dose--response curve to ET-1. In contrast, at a concentration of 10(-10) M, rANF 99-126 but not rANF 103-126 produced a significant leftward shift of the dose--response curve to ET-1 and an increase in the maximal developed tension for the dose--response curve to ET-1. For tissues incubated in the absence of extracellular calcium or in the presence of the calcium channel blocker nifedipine (5 x 10(-7) M), both ANF derivatives produced a dose-dependent decrease in the maximum contraction, but no change in potency to ET-1. Addition of either rANF 103-126 or rANF 99-126 to tissues maximally contracted with ET-1 resulted in relaxation, reaching a maximum of 70%. The ED50 values for relaxation were 2.7 +/- 0.51 x 10(-8) and 3.5 +/- 0.60 +/- 10(-8) M for rANF 103-126 and rANF 99-126, respectively. ET-1 did not interact with biologically responsive and clearance receptors for ANF.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue specificity of endothelin binding sites.

A measurement was made of the binding of 125I-labeled endothelin (125I-ET) to crude membrane fractions prepared from rat aorta, atrium, ventricle, portal vein, trachea, lung parenchyma, vas deferens, ileum, bladder, and guinea-pig taenia coli and lung parenchyma. Scatchard analysis of 125I-ET binding in all tissues indicated binding to a single class of saturable sites. The affinity and density of 125I-ET binding sites varied between tissues. The Kd of 125I-ET binding was approximately 0.5 nM for rat aorta, trachea, lung parenchyma, ventricle, bladder, and vas deferens, and guinea-pig taenia coli and lung parenchyma, 1.8 nM for rat portal vein and atrium, and 3.3 nM for ileum. The Bmax of 125I-ET binding had the following rank order of density in rat tissues: trachea greater than lung parenchyma = vas deferens much greater than aorta = portal vein = atrium greater than bladder greater than ventricle = ileum. The properties of 125I-ET endothelin binding were characterized in rat ventricular membranes. 125I-ET binding was time dependent, reaching a maximum within 45-60 min at 25 degrees C. The calculated microassociation constant was 9.67 x 10(5) s-1 M-1. Only 15-20% of 125I-ET dissociated from its binding site even when dissociation was studied as long as 3 h. Preincubation of ventricular membranes with ET prevented binding of 125I-ET. 125I-ET binding was destroyed by boiling of ventricular membranes and was temperature, pH, and cation (Ca2+, Mg2+, and Na+) dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-lipotropin-like immunoreactivity in intraglandular colloid from pituitary intermediate lobe cells.

The aim of this paper is the identification of beta-lipotropin (beta/LPH) as a peptide present in intraglandular colloid (the holocrine secretion of cells in the marginal half of the bovine pituitary intermediate lobe). beta/LPH, although not an opioid peptide itself, contains the peptide beta-endorphin. The methodology used allowed detection of beta/LPH when present in the samples in sufficient amounts.

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12.

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.

Presence of arginine-vasopressin in bovine pituitary intraglandular colloid of intermediate lobe origin.

In order to determine whether pituitary intraglandular colloid might contain enough hormonal material to be considered a transport medium for intermediate lobe peptides, arginine-vasopressin was assayed in three pools of colloid collected from nearly 100 steers. Colloid samples taken from the pituitary glands of freshly slaughtered cattle were pooled, freed of extraneous tissue, and lyophilized. Three such pools were further extracted on octadecylsilyl-silica and assayed. The radioimmunoassay showed that arginine-vasopressin was indeed present in the bovine intraglandular colloid at levels ranging from 0.18 to 6.95 ng/mg dry weight. These results indicate that the intraglandular colloid contains a sufficient amount of arginine-vasopressin to be considered as a transport medium for this and other intermediate lobe peptides.

Presence of alpha-melanocyte-stimulating hormone in bovine pituitary intraglandular colloid of intermediate lobe origin.

Alpha-melanocyte-stimulating hormone (alpha-MSH), one of several peptide hormones originating in the intermediate lobe of the pituitary as proopiomelanocortin, was discovered in bovine pituitary intraglandular colloid by using radioimmunoassay. The quantity of alpha-MSH varied from 5 to 368 micrograms/mg protein in the three pools. The importance of this finding is discussed in light of the possibility that the colloid is a transport medium for alpha-MSH and other intermediate lobe hormones.

Presence of met-enkephalin in bovine pituitary intraglandular colloid of intermediate lobe origin.

Met-enkephalin, and opioid peptide present in the intermediate lobe of the pituitary gland, is herein demonstrated, for the first time, in bovine pituitary intraglandular colloid. Colloid samples were taken from the glands of freshly slaughtered cattle, pooled, lyophilized, extracted on octadecylsilyl-silica and assessed by radioimmunoassay. Met-enkephalin was indeed present in this colloid extract at levels varying from 0.40 to 0.85 nanograms per milligram dry weight. Locating an intermediate lobe peptide in intraglandular colloid is a significant finding since this implies that colloid, housed in the intraglandular lumen, may serve as a transport medium. Intermediate lobe peptides which, because the intermediate lobe is avascular, are otherwise denied the vascular transport routes available to the anterior and posterior lobe hormones.

Proline transport and osmotic stress response in Escherichia coli K-12.

Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.

ACTH in bovine pituitary intraglandular colloid, the holocrine secretion of intermediate lobe cells.

The presence of bovine pituitary intermediate lobe peptides in intraglandular colloid, the holocrine secretion of intermediate lobe cells, is explored by ELISA. Intraglandular colloid collected immediately after sacrificing the animal, is placed in phosphate buffered saline, pH 7.6. This material is homogenized, centrifuged to remove extraneous tissue, lyophilized and stored at -20 degrees C. ACTH in intraglandular colloid is measured by competitive ELISA. Human ACTH (1-24) is used in the preparation of the solid phase antigen and as the standard for competition. The antibody is rabbit anti-human ACTH (1-24), and the alkaline phosphatase conjugate is goat anti-rabbit IgG with p-nitrophenyl phosphate as substrate. It is concluded that ACTH is present in bovine pituitary intraglandular colloid of intermediate lobe origin and that the colloid may serve as a transport medium for intermediate lobe materials.

Detection of regulatory receptors and distinctive surface markers.

Cell sorting has been used as an approach to describing surface markers and functional receptors of hemopoietic stem cells and progenitors. Significant enrichment of CFU-S was obtained with fluorescent cell sorting of human antihuman sperm-labelled bone marrow cells; significant enrichment of human CFU-C was obtained by cell sorting of hybridoma (KGP1)-labelled human bone marrow cells. Significant changes in human bone marrow CFU-C, but not CFU-E were observed by fluorescence polarization after exposure to a putative stimulator of CFU-C. A description of preliminary investigations of a hybrid clone from Dexter culture cells x Chinese hamster ovary and fluorescence polarization measurements after exposure to purified mouse-lung CSF are also presented.

Hemopoietic stem cells bear sperm-associated antigens.

A fluorescence-activated cell sorter has been used to sort out enriched populations of murine hemopoietic stem cells from mouse bone marrow, based on fluorescence of cells labeled with various antisera. Significant positive enrichment for stem cells was obtained with rabbit anti-human brain, human anti-human sperm and mouse anti-mouse F9 sera but not with mouse anti-mouse Ia serum. These results support the hypothesis that the hemopoietic stem cell exhibits 'unique' surface antigens cross-reactive with sperm, brain and embryonic antigens.

Characterization of stem cells and progenitors of hemopoiesis by cell sorting.

Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).

Serologic identification of hemopoietic progenitor cell antigens common to mouse and man.

An immunologic approach to the identification of human hemopoietic stem cell antigens has yielded two types of antisera. Both recognize antigens on hemopoietic progenitors which are identical or cross-reactive with determinants on sperm, brain, and, across the species barrier, with murine hemopoietic stem cells (CFU-S). The first antiserum was the result of an autoimmune reaction in a post-vasectomy patient. The second was produced in rabbits after injection of human brain cells.

Age-related changes in T cell function.

A comparison was made of the abilities of carrier (BGG)-primed T cell populations from young (4-month old), middle-aged (14- and 19-month old) and old (31- and 34-month old) mice to collaborate with hapten (DNP)-primed B cells from young mice in a cell-transfer system. The plaque-forming cell responses to 2,4-dinitrophenol (DNP) were measured by a modification of the Jerne plaque assay. The DNP-specific antibody-forming cell responses of old T cell/young B cell combinations were significantly lower than those of young T cell/young B cell combinations, both in the number of T cells needed for peak response and in the size of that response. These data indicate that the primed T cell populations of old mice are deficient by a factor of 6 in their ability to initiate B cell proliferation and differentiation into antibody-forming cells.