Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes

Human non-secretory neutral ribonucleases (RNases) from kidney,liver and spleen have been purified and characterized. SDS—PAGEindicates that all three RNases are highly purified and haveapparent mol. wts of 17–18 kDa. Kinetic analysis indicatesthat all three RNases have a broad pH optimum centred around6.5, and all three have similar substrate specificities withsignificant preference for RNA and poly(U) when compared topoly(C), poly (A) and poly(G). All of the above data, as wellas immunoblotting data using three polyclonal antibodies (anti-humanliver RNase, anti-human pancreatic RNase, anti-human eosino-phil-derivedneurotoxin), indicate that the three proteins are highly purifiedand are non-secretory RNases (IIN). Further characterizationby cyanogen bromide peptide mapping and extensive lectin blottingindicated no significant differences between the three humanRNases. All three RNases appear to have very similar, if notidentical, protein backbones and all three are glycoproteinswhich are recognized by lectins with specificity for GlcNAc,Fuc and, to a lesser extent, with specificity for Galß(1–4)GlcNAc.No significant tissuespecific differences were found among thethree human non-secretory RNases. lectin blotting non-secretory RNases peptide mapping     

Quantitation of doxapram in blood,plasma and urine

Methods for the quantitation of doxapram in blood, plasma and urine have been developed. Following extraction, gas—liquid chromatography was used to separate doxapram from basic metabolites. Doxapram was detected by mass spectrometry for blood and plasma assays, and by flame ionisation for urine assays. The limit of reliable quantitation in blood and plasma was 10 ng and in urine 500 ng, the coefficients of variation being 6.37%, 1.72% and 2.31% respectively. To illustrate the clinical applicability of the assay methods, plasma, blood and urine levels were monitored in a premature newborn following an intravenous infusion of doxapram.     

Purification and characterization of three ribonucleases from human kidney: comparison with urine ribonucleases

Three ribonucleases (RNases) with different molecular masses were isolated from human kidney. The enzymes were purified to an electrophoretically homogeneous state, and their respective molecular masses were found to be 18,000 (tentatively named RNase HK-1), 20,000 (RNase HK-2A), and 22,000 (RNase HK-2B) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the amino acid compositions, amino-terminal sequences, and enzymological properties of the enzymes indicate that RNase HK-1 is related to "nonsecretory" RNase, and that RNases HK-2A and HK-2B are both related to "secretory" RNase. Furthermore, RNase HK-1 showed cross-reactivity with an antibody specific to nonsecretory RNase from human urine, whereas RNases HK-2A and HK-2B showed cross-reactivity with another antibody specific to human urine secretory RNase. However, the carbohydrate compositions of RNases HK-2A and HK-2B were markedly different from that of the secretory urine RNase. This finding seems to indicate that the kidney is not the origin of the urine enzyme.     

Human non-secretory ribonucleases. II. Structural characterization of theN-glycans of the kidney, liver and spleen enzymes by NMR spectroscopy and electrospray mass spectrometry

The N-glylycans have been removed by peptide-N-glycosidase F(PNGase F) from purified human non-secretory RNases derivedfrom kidney, liver and spleen. The spleen RNase was purifiedby two procedures, one of which did not include the usual acidtreatment step (0.25 M H₂SO₄, 45 min, 4C), to determine ifacid treatment alters the carbohydrate moieties. TheN-glycansof the RNases were fractionated by Bio-Gel P-4 chromatographyand analysed by 600 MHz ¹H-NMR spectroscopy and electrospraymass spectrometry. All four non-secretory RNase preparationscontained the following structures: The relative amounts of the trisaccharide, pentasaccharide andhexasaccharide appeared to vary slightly in the different tissueRNases. The overall results indicate: (i) that acid treatmentduring purification does not alter the N-glycans of non-secretoryRNases; (ii) that the N-glycans from kidney, liver and spleennon-secretory RNases are very similar, if not identical, toone another, but different from the N-glycan structures reportedfor secretory RNase. N-glycans non-secretory RNases     

N-acetyl-beta-d-glucosaminidase in marmoset kidney, serum and urine.

N-Acetyl-beta-D-glucosaminidase activities were determined in homogenates of marmoset kidney, in serum and in urine by using the 4-methylumbelliferyl substrate. The enzyme activity was separated into several components by DEAE-cellulose ion-exchange chromatography, starch-gel electrophoresis and isoelectric focusing. The kidney contained two major forms of the enzyme, A and B, which had similar pH optima and Km values. The A-form bound to DEAE-cellulose at pH 6.8, migrated towards the anode on starch-gel electrophoresis and had a pI of 5.0. The B-form did not bind to DEAE-cellulose at pH 6.8, remained near the origin on starch-gel electrophoresis and had a pI of 7.64. The isoenzymes also differed in heat stability, the B-form being the more stable. Serum contained B-form activity and, in addition, two intermediate forms (I1 and I2) were loosely bound to DEAE-cellulose. The serum A-form activity was less firmly bound to DEAE-cellulose than was the tissue A-form and was designated As. Serum from a pregnant marmoset contained a form which may be analogous to the human P-isoenzyme. Urine contained only a small amount of B-form activity, the majority being present in the A-form. The kidney A- and B-forms both had mol.wts. of 96000--100000 and the activity was predominantly lysosomal. Partial purification of the kidney A isoenzyme was undertaken. Immunoprecipitation studies indicated a relationship between marmoset kidney A-form and human liver A-form activity.     

Isolation and characterization of two alkaline ribonucleases from calf serum.

Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A. The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate.     

Studies on prolactin in human serum, urine and milk.

Prolactin activity was measured in serum, urine and milk using a specific human prolactin radioimmunoassay (RIA). Serum, urine and milk were parallel with the human prolactin standard in the RIA. There was no correlation between serum prolactin levels and urinary prolactin activity. Dialysis of urine samples resulted in complete loss of human prolactin activity while the addition of human prolactin to the urine resulted in the recovery of over 50% of the hormone after dialysis. Thus it was concluded that prolactin is not present in urine. In additional experiments it was observed that the RIA prolactin activity in urine was significantly correlated with the osmolality of the urine and that Na+ and K+ were contributory elements. On the other hand, prolactin was found in human milk and correlated well with the expected serum levels of this hormone. This latter finding is interesting because prolactin receptors have been shown to exist on the serosal side of the mammary epithelial cells. The presence of prolactin in milk suggests the possibility of other sites of action for this hormone in addition to the cell membrane.     

Discovery of "masked" ribonucleases in highly purified preparations of RNA

It was found that RNA preparations isolated with the help of various inhibitors of RNAses from different eukariotic tissues, followed by thorough deproteinization contain particular ribonucleases ("masked" RNAses). These RNAses were supposed to be connected with RNA molecules and are not active. They may be activated by changes of RNA molecule conformation. Apparently the "marked" RNAses can take part in (a) processing of large precursor RNA molecules; (b) regulation of gene expression by means of specially cut RNA fragments.     

Human serum deoxyribonuclease I (DNase I) polymorphism: pattern similarities among isozymes from serum, urine, kidney, liver, and pancreas.

We have devised a zymogram method with high sensitivity and resolution for investigating molecular heterogeneity and genetic polymorphism of deoxyribonuclease I. A combination technique of polyacrylamide-gel isoelectric-focusing electrophoresis and the newly developed zymogram method have led to the discovery of genetic polymorphism of human serum DNase I. Family studies showed that the three common phenotypes--DNASE1 1, DNASE1 1-2, and DNASE1 2--and the other five relatively rare phenotypes--DNASE1 1-3, DNASE1 2-3, DNASE1 2-4, and DNASE1 3-4--represent homozygosity or heterozygosity for four autosomal codominant alleles, DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4. The frequencies of DNASE1 *1, DNASE1 *2, DNASE1 *3, and DNASE1 *4 calculated in a Japanese population were .5517, .4358, .0104, and .0021, respectively. Moreover, it was found that urine and extracts of kidney, liver, and pancreas, as well as serum, can be used for DNase I phenotyping.     

Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells

The distribution of membrane-bound receptors and enzymes between the cell surface and the cell interior can be determined without solubilization or gross disruption of cell organelles in the presence of the nonionic detergent digitonin. This steroid glycoside permeabilizes cells, releases cytoplasmic proteins with subunit molecular weights up to 200,000, and allows exogenous molecules to gain access to intracellular receptors. All cell types examined were affected similarly by digitonin. Permeabilization was complete within 2 min at 0°C and did not require the continued presence of digitonin. A characteristic amount of protein (～50%) was lost between 0.02 and 0.08% ($\text{w}\text{v}$) digitonin. Three independent systems were examined: the insulin receptor in 3T3 fibroblasts and the asialoglycoprotein receptor and the $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}\text{-ATPase}$ in rat hepatocytes. In each case an increase in the specific activity of enzyme/receptor occurred over a range of detergent concentration in which the retention of cell protein was constant and virtually no solubilization of membrane-bound activity occurred. The binding of ¹²⁵I-asialo-orosomucoid to rat hepatocytes at 0°C in the presence of digitonin was linear with cell number and kinetically indistinguishable from binding to intact cells. Receptors exposed by digitonin were shown to be intracellular by light microscopic examination of permeabilized cells first treated with antiserum to the receptor and then with a second antibody horseradish peroxidase conjugate. The use of digitonin has many advantages over procedures which require total cell disruption or solubilization to assess intracellular receptors. The technique has already been valuable in studies on recycling and endocytosis mediated by the asialoglycoprotein receptor (P. H. Weigel and J. A. Oka (1983)J. Biol. Chem.258, 5095–5102) and should also be useful in studies with other membrane-bound receptors and enzymes in other cell types.     

Human epidermal growth factor concentrations in urine, but not in saliva and serum, depend on thyroid state

To evaluate the effects of thyroid hormones on the concentration of epidermal growth factor (EGF), we determined values for the immunoreactive EGF concentration in the urine (U-irEGF) of newborn infants with congenital hypothyroidism (N = 19), and in urine, saliva and serum of adult patients with hypothyroidism (N = 11) and hyperthyroidism (N = 8). The values were expressed as SD score (SDS), i.e. deviation in SD units from their mean value of healthy subjects of the same age and sex. The SDS of relative U-irEGF (ng/mg creatinine) was lower (P less than 0.01) in newborn infants with congenital hypothyroidism (-0.8 +/- 0.2; mean +/- SEM) than in healthy infants. Their relative U-irEGF correlated with their serum T4 concentrations (r = 0.59, P less than 0.01). The SDS of relative U-irEGF was lower (P less than 0.01) in adult hypothyroid patients (-1.2 +/- 0.5) and higher (P greater than 0.05) in adult hypothyroid patients (0.9 +/- 0.6) than in healthy adult subjects. When subsequently euthyroid, their SDS of relative U-irEGF increased to -0.5 +/- 0.3 (P less than 0.01), and decreased to -0.7 +/- 1.1 (P less than 0.05), respectively. The irEGF concentrations in saliva and serum were not significantly different between the hypothyroid and hyperthyroid patients. Our results indicate that urinary excretion of irEGF in man is dependent on thyroid hormone.     

Parathyroid hormone preparations, salmon calcitonin, and urine flow in the South American lungfish, Lepidosiren paradoxa.

In the South American lungfish, Lepidosiren paradoxa, injections of salmon calcitonin or bovine parathyroid extract have no effect on serum calcium levels. However, salmon calcitonin elicits a distinct diuresis. Crude or purified parathyroid extract or synthetic parathyroid hormone (1-34 fraction) produce a distinct antidiuresis.     

Quantitation of human anti-mouse antibody in serum by flow cytometry.

Clinical investigations utilizing murine monoclonal antibodies require techniques for the detection of the human anti-mouse antibody (HAMA) response in patient serum. We report here a flow cytometric assay for the quantitation of HAMA. Commercially available beads conjugated with goat anti-mouse antibody provide a solid phase matrix for a triple bridge immunoassay. The measurement of fluorescein labeled antibodies by flow cytometry allows accurate quantitation of the HAMA. The assay will detect antibody levels of approximately 1.0 ng/ml. Antibody recovery in serum samples with known amounts of antibody added was greater than 90% at levels greater than or equal to 10 micrograms/ml. Serum samples obtained from 41 patients prior to and after single or multidose infusions of KS1/4-Desacetyl-vinblastine were analyzed. These results were compared with HAMA titers previously determined by ELISA. With few exceptions, patients with low titers as determined by ELISA demonstrated low HAMA potencies by flow cytometry and those with highest titers the highest potencies. Patients with no detectable HAMA by ELISA were also negative by flow analysis. The results of our studies demonstrate that HAMA levels can be accurately and quantitatively determined by flow cytometry.     

Specific elimination of albumin in serum lipoprotein preparations.

In the preparation of pure serum lipoproteins by means of ultracentrifugation techniques, it is necessary to perform consecutive centrifugations in order to reduce the content of contaminating serum albumin (1) impairing apolipoprotein determination and purification and turnover studies with ¹²⁵I-labeled lipoproteins. Extensive centrifugation, however, may cause in vitro changes in the composition of the lipoprotein particles (1,2). The present report describes the use of matrix-bound anti-albumin for specific elimination of albumin in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) preparations from human serum.