Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

Relationships between Biovolume and Biomass of Naturally Derived Marine Bacterioplankton

Microscopic estimation of bacterial biomass requires determination of both biovolume and biovolume-to-biomass conversion. Both steps have uncertainty when applied to the very small bacteria typically found in natural seawater. In the present study, natural bacterioplankton assemblages were freshly collected, passed through 0.6-μm-pore-size Nuclepore filters to remove larger particulate materials, and diluted for growth in 0.22-μm-pore-size Millipore filter-sterilized unenriched seawater. This provided cells comparable in size and morphology to those in natural seawater, but the cultures were free of the interfering particulate detritus naturally present. Cells were collected on glass-fiber GF/F filters, and biovolumes were corrected for cells passing these filters; C and N were measured with a CHN analyzer. Our criteria for size measurement by epifluorescence photomicrography were confirmed with fluorescent microspheres of known diameters. Surprisingly, in six cultures with average per-cell biovolumes ranging from 0.036 to 0.073 μm³, the average per-cell carbon biomass was relatively constant at 20 ± 0.08 fg of C (mean ± standard error of the mean). The biovolume-to-biomass conversion factor averaged 0.38 ± 0.05 g of C cm⁻³, which is about three times higher than the value previously estimated from Escherichia coli, and decreased with increasing cell volume. The C:N ratio was 3.7 ± 0.2. We conclude that natural marine bacterial biomass and production may be higher than was previously thought and that variations in bacterial size may not reflect variations in biomass per cell.     

The sugar-specific adhesion/deadhesion apparatus of the marine bacterium Vibrio furnissii is a sensorium that continuously monitors nutrient levels in the environment

Our earlier studies on cell adhesion to immobilized carbohydrates are extended here to a marine bacterium, Vibrio furnissii. Apparently one lectin mediates the binding of these cells to glycosides of N-acetylglucosamine, mannose, and glucose covalently linked to Agarose beads. Kinetic studies show that protein synthesis is required for initiating and for maintaining adhesion to the glycosides. Furthermore, a pro- mutant binds to GlcNAc-beads at Pro concentrations insufficient to support cell growth. Expression of the functional lectin therefore predominates under conditions of limiting protein synthesis. Thus, cells adhere to the sugars in an environment compatible with protein synthesis, and deadhere when depleted of any required nutrient, presumably to migrate to a more favorable locale. The adhesion-deadhesion apparatus thereby permits constant monitoring of the surrounding environment, comprising a "nutrient sensorium".     

Solubilisation and reconstitution of acylcoenzyme A:estradiol-17 beta acyltransferase

Acylcoenzyme A:estradiol-17 beta acyltransferase in microsomes of bovine placenta cotyledons was strongly membrane bound. The enzyme was solubilised from microsomes by sodium cholate and was reconstituted into phospholipid vesicles. The apparent Km for estradiol-17 beta was 11 microM which was close to the value of 8 microM previously found with the membrane-bound enzyme. Testosterone was also a substrate for the reconstituted enzyme (apparent Km 62 microM) and was a competitive inhibitor (Ki 74 microM) of the acylation of estradiol-17 beta. Although various long-chained fatty acyl CoAs acted as acyl donors, these proved to have widely differing apparent Km values with palmitoleoyl CoA having the highest affinity (Km 24 microM) and arachidonoyl CoA the lowest affinity (Km 330 microM).     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.     

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.     

Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels

We present a new method for visualizing proteins electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are incubated in CuCl2 to produce a negative image of colorless protein bands against a semiopaque background. Gels are stained completely within 5 min, do not require destaining, and can be stored indefinitely without loss of the image. Because proteins are not permanently fixed within the gel, they can be quantitatively eluted after chelation of Cu with EDTA. The sensitivity of the CuCl2 stain falls between that of Coomassie blue and silver. We anticipate that CuCl2 will be useful in the rapid analysis of proteins by polyacrylamide gel electrophoresis and in the preparation of purified polypeptides by elution from gel slices.     

Thymine glycol-DNA glycosylase/AP endonuclease of CEM-C1 lymphoblasts: a human analog of Escherichia coli endonuclease III

A thymine glycol-DNA glycosylase/AP endonuclease has been identified in human CEM-C1 lymphoblasts. The enzyme is active in the absence of divalent cations and has an apparent molecular size of approximately 60,000 daltons. The enzyme releases thymine glycol from osmium tetroxide-damaged DNA via an N-glycosylase activity and is associated with an endonuclease activity that mediates phosphodiester bond cleavage at sites of thymine glycol and apurinic sites. We propose that this enzyme, which we call redoxyendonuclease, is the human analog of a bacterial enzyme, E. coli endonuclease III, that recognizes oxidative DNA damage.     

Detergents as probes of reconstituted rat liver cytochrome P-450 function

A series of 16 ionic, zwitterionic, and nonionic detergents have been used to perturb the catalytic activities of major cytochrome P-450 (P-450) forms from untreated (UT-A), phenobarbital-treated (PB-B) and beta-naphthoflavone-treated (BNF-B) rats in reconstituted systems with NADPH--P-450 reductase Detergent effects on R warfarin hydroxylase activities were correlated with detergent effects on the quaternary structures of P-450 and reductase, and on their 1:1 complexes as determined by gel exclusion chromatography using sodium cholate as a prototype detergent. The detergent concentrations used did not in most cases affect rates of NADPH-dependent reduction of cytochrome c by the reductase. With P-450 BNF-B, ionic and zwitterionic detergents enhanced warfarin hydroxylase activities at low concentrations and produced marked inhibition at higher concentrations, while nonionic detergents only inhibited. With P-450 UT-A, some nonionic and zwitterionic detergents increased rates at low concentrations and inhibited at higher concentrations. P-450 PB-B was inhibited by detergents of all three classes at low and high concentrations. The concentrations of a detergent required to affect 50% inhibition differed for the three P-450s, suggesting, together with the differential susceptibilities to detergent-mediated rate enhancing effects, that the reductase interacts functionally differently with the three P-450s. Chromatographic studies demonstrated that concentrations of sodium cholate which optimally enhanced metabolic rates with P-450 BNF-B facilitated the uptake of the P-450 into the functional reductase/P-450 complex, and higher concentrations of cholate, which completely inhibited activity, produced profound disruptions of the complex. The data have provided insight into the functional interactions required for monooxygenase activity.     

Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)