A comparison of four preparations of erythropoiesis regulatory factors

BIOCHEMICAL

MEDICINE

14, 399410

(1975)

A Comparison of Four Preparations of Erythropoiesis Regulatory Factors’ J. P. LEWIS, EMILY T. WELCH, W. A. NEAL, C.-S. WRIGHT, E. GARDNER, JR., LINDA L. SMITH, AND R. D. LANGE Department

of Research, Veterans Administration Hospital. Augusta, Georgia Departments of Medicine and Cell and Molecular Biology, Medical College of Georgia. Augusta, Georgia 30902, and University of Tennessee. Memorial Research Center and Center for the Health Sciences. Knoxville. Tennessee 37920

30904,

Received January 5, 1976

The properties of an erythropoietin-generating factor (EGF), obtained from the urine of anemic patients, have been reported by Gardner et ~1. (1,2). Earlier, Kuratowska et al. had described the properties of an erythropoietin-generating substance obtained from the perfusates of isolated anoxic kidneys (3). Gordon rt al. confirmed and extended the study of generating factor(s) in anoxic kidneys and called their factor erythrogenin (4). EGF was demonstrated to have a molecular weight between 30,000 and 50,000 and to be closely associated with an erythropoietin (ESF,) preparation, which had a molecular weight between 20,QOO and 30,000 (5). Dorado et al. showed that it was possible to separate erythropoietic activity into several components by polyacrylamide-gel electrophoresis and electrofocusing (6). Reelectrophoresis of the separated fractions then resolved the ESF activity into one peak. When dodecylsulfate (SDS)-erythropoietin complexes were formed, Espada found that the ESF components moved as one single entity during electrophoresis or chromatography (7). Dorado et al. reported the molecular weight of erythropoietin to be 23,000, as determined by SDS-polyacrylamide-gel electrophoresis (8). Dukes et al. found a regulatory protein associated with erythropoietin that modified its dose response (9). The purpose of this report is to compare EGF and ESFr , obtained from a female patient with an anemia secondary to multiple myeloma (MM) and a male patient with paroxysmal nocturnal hemoglobinuria (PNH), with the 1 Supported by Veterans Administration Hospital (MRIS 5224-03), Medical College of Georgia (NIH grants, No. FR-5365, FR-0061, and HE-12958), and University of Tennessee, Memorial Research Center (USPHS Grant No. HE-10567). We wish to acknowledge the technical assistance of Mrs. Carol Cope, Miss Betty Williams, Mrs. Yvonne Quirin, and Mrs. Betty Newton. 399 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.

400

LEWIS ET AI..

erythropoiesis regulatory factors(s) (ERFs) from the urine of patients with anemia caused by hookworm infestation.2 MATERIALS

AND

METHODS

The ERF pool sent to the VA Hospital in Augusta, H- IO-Ta LSL (NIHERF), was made from three individual batches of concentrates of erythropoietin prepared by the benzoic acid adsorption method of Espada and Gutnisky (10). This material was solubilized and purified by extraction into water (including a 45set exposure to 60°C temperature), Iyophilization. Sephadex G-25 gel chromatography to remove salts and pigments, and re-lyophilization. Based upon the “exhypoxic polycythemic mouse-jYFe incorporation” assay, the specific activity of Pool H-IO-Ta LSL material was estimated to be 75.4 ? 5.8 IU/mg as determined by the assav by Dukes et al. (11). The units were based on the WHO International Reference Preparation. The NIH-ERF was dissolved in 0.02 M phosphate buffer, pH 7.4,0.5 M NaCl and incubated for 30 min at 37”C, so that each assay mouse would receive 0.38 IU of NIH-ERF. A similar solution was prepared except that 10 PM dithiothreitol (DTT) was included in the incubation mixture. Both solutions were injected intraperitoneally (ip) into exhypoxic-polycythemic mice (12). The assay results are reported as the percentage of red blood cell 5YFeincorporation during 48 hr and also as international standard3 B units. A solution calculated to contain 0.68 IU/ml in 0.02 M phosphate buffer, pH 7.4, in 0.5 M NaCl was prepared, and 13.7 ml was fractionated with Amicon XM-25 centriflo membranes supported in 50-ml conical centrifuge tubes. These membranes are designed to retain molecules of a molecular weight of about 25,000 or greater. Centrifugation at 4°C was continued at 1 1OOg until all the solution had passed through the membrane. The filtrate was collected and the residue on the membrane was dissolved in an amount of various buffers equal to the volume of the filtrate. Aliquots of the filtrate and the latter solution (referred to as the retentate) were injected ip into exhypoxic-polycythemic assay mice. The 0.02 M buffers used with an equivalent of about 0.25 IU of ESF (NIH-ERF) per mouse of the residue were: sodium acetate-acetic acid, disodium and monopotassium phosphate, tris(hydroxymethyl)amino methane-HCl, and sodium borate-boric acid. Normal rabbit serum was added to the NIH-ERF. and the mixture was ’ The latter urine was collected and concentrated by the Department of Physiology. University of the Northeast, Corrientes, Argentina, and then further processed and assayed by the Hematology Research Laboratories, Children’s Hospital of Los Angeles (Research Grant No. HE-10880, National Heart and Lung Institute). R Standard B was obtained through the courtesy of Drs. D. K. Bangham and Mary Cotes, Medical Research Council. National Institute for Medical Research. London. England.

ERYTHROPOIESIS

REGULATORY

FACTORS

401

incubated at 37°C for 1 hr. The incubated mixtures and the NIH-ERF fraction were each injected ip into assay mice. Strain AKR female mice made polycythemic by exposure to a simulated altitude of 7320 m were used at the VA Hospital in Augusta. The mice were intermittently kept in glass enclosures in an atmosphere of 8% oxygen and 92% nitrogen (12). The packed cell volumes were determined by a microhematocrit technique. The mice had a mean packed red blood cell volume of 55.6 + 0.2% at the conclusion of the assay (13). Mice injected with 0.9% saline solution were used as controls with all groups. The values for the saline solution-injected control groups were subtracted from the ESF activity values, which had been determined from a log dose-log response curve. Ten mice were used for each assay group. Immunizations were done as reported by Lange et al. (14). Eight rabbits were immunized with an EGF fraction obtained from the urine of a patient with MM, and a second group of eight rabbits was immunized with an EGF fraction from the urine of a patient with PNH (15). One out of eight of the rabbits immunized with the EGF fraction from the PNH urine produced an EGF-neutralizing antibody, and that is the antiserum used in the work described herein. The remainder of the rabbits produced non-neutralizing but precipitating antibodies. A similar response was obtained with eight rabbits immunized with the EGF from the MM urine, except that the one rabbit produced a relatively weak neutralizing antibody. A control containing 0.05 mg of the EGF fraction was incubated in phosphate-buffered saline (PBS) for 30 min at 37°C. The EGF fraction (0.05 mg) was also incubated with 0.2 ml of anti-EGF antiserum. The described experiment was done in duplicate. Additional duplicate experiments were done identically except that the solutions were centrifuged and the supernatant fluids were injected into the assay mice. Assays demonstrating an anti-ESF, neutralizing antiserum were done at the Medical College of Georgia with CD- 1 mice. The assay mice’were made polycythemic by enclosure in silicone membrane cages as described by McDonald and Lange ( 16). An ESF, fraction was used to immunize a group of seven rabbits as previously reported (15). Five rabbits in the group receiving ESF, responded by producing neutralizing antibodies to erythropoietin. The ESF,-neutralizing antibodies were used in the work described herein. Controls containing either the ESF, fraction or the EGF fraction (each containing sufficient material to inject 0.5 mg/mouse) were incubated in saline for 30 min at 37°C. Another incubation mixture contained various amounts of rabbit anti-ESF, antiserum in addition to 5.0 mg of the ERF fractions for 10 mice. NIH-ESF (0.16 IU) from Pool H-6-TaLSL (36.0 IU ESF/mg), sent to the University of Tennessee Memorial Research Center in Knoxville, was

402

LEWIS

ET AL

incubated with 0.2 ml of anti-EGF antiserum at 37°C for 30 min and then assayed in exhypoxic-polycythemic mice. The experiment was repeated except that the solution was centrifuged, and the supernatant fluid was injected into the assay mice. Strain C,H/He female mice were made polycythemic by hypoxia with a modified technique (17) of the method described by McDonald and Lange (16). The mice were kept in silicone rubber membrane enclosures. The results are reported as the percentage of jsFe incorporated into RBC during 48 hr and as international units of ESF obtained from a log dose-log response curve. Eight mice were used for each assay group. Polyacrylamide-gel electrophoresis (PAGE) was used to characterize the EGF from the urine of the patient with MM (18,19). The neutral pH-SDS-gel system was used as described by Maize1 (20). The gels were composed of 7.5% acrylamide, 0.2% N.N’-methylene-bisacrylamide, 0.1 M Na,PO, buffer, pH 7.2, 0.1% SDS, 0.5% N,N.N’. N’-tetramethand 0.1% ammonium persulfate. The acrylamide ylenediamine, solution was allowed to polymerize in 65 x 6-mm tubes. The running buffers were 0.1 M phosphate, pH 7.2, and 0.1% SDS. Samples containing protein, 0.1 mg for EGF and as much as 2 mg for standards, were applied to the top of the gels in 50% sucrose solution with and without bromophenol blue. Electrophoresis was done at 7.5 mA per tube. After electrophoresis the gels were stained with periodic acid-Schiff (PAS) stain by the methods of Glossman and Neville (21). Comparable PAGE experiments were done with up to 100 mg of an EGF fraction. The stained gels were scanned at 550 nm and unstained gels at 280 nm in a Gilford 240 spectrophotometer equipped with a linear transport system. The proteins IgG (MW lSS.OOO), serum albumin (MW 67,000), and ovalbumin (MW 45,000) were used as standards. With a semilog plot of the molecular weight of the standards vs the distance migrated, the molecular weight of the unknown protein was determined. The gels were sliced into S-mm pieces, and the proteins of the EGF fraction were eluted and tested for the capability of producing ESF activity. RESULTS

When 0.38 IU of NIH-ERF was incubated as described and injected into each of 10 mice,-the percentage of 5sFe incorporation was 15.46 + 0.22. When the same amount of NIHERFwas incubated with 10 PM DTT prior to injection as described, the percentage of 5s‘Fe incorporation in the red blood cells was 4.87 ? 0.42 (Table 1). When a solution of NIH-ERF containing an estimated 0.68 IU of ESF activity/ml in 13.7 ml was filtered thru Amicon XM-25 Centriflo membranes as described, the ESF activity in the filtrate was 11.25 ? 0.27% ssFe/ml uptake in RBC; when the residue on the membrane was dissolved in the

ERYTHROPOIESIS

TREATMENTOF AKRMIcE 10 PM DITHIOTHREITOL THROUGH

Treatment per mouse with NIH-ERF A. Incubated NIH-ERF 0.38 IU 0.38 IU with DTT B. Filtered NIH-ERF Retentatec, 1 ml Filtrate, 1 ml

REGULATORY

403

FACTORS

TABLE 1 NIH-ERF(s)” INCUBATEDWITHOUTANDWITH (DIT) AND FILTRATION OF NIH-ERF SOLUTION AMICON XM-25 CENTRIFLO CONES

WITH

Percentage of ssFe incorporated in RBC(kSEM)

ESF activity (IU f SEM)

Percentage of total activity

15.46 2 0.22 4.87 2 0.42 (Inactivated ERF)-+

0.34 k 0 0.10 f 0.01 0.24

100 29.4 70.7

0.62 + 0.01 0.24 k 0.01

72.2 27.8

25.99 +- 0.37 11.25 f 0.27

n NIH-ERF(s) = erythropoiesis regulatory factors distributed by the National Heart and Lung Institute. * NIH-ERF solutions were prepared as described in Materials and Methods. The initial activityof0.38IWml was baseduponasolution(5.0 x lo-smg/ml)oftheNIH-ERFwhich had an assay value of 75.4 f 5.8 IU/mg, as reported from the Hematology Research Laboratories, Children’s Hospital of Los Angeles. Our assay (in Augusta) for that solution was 0.34 k 0 IUlml. A solution of 0.68 W/ml was used in experiment B, since ESF, was expected in the filtrate and EOF rn the retentate. c Retentate is the residue redissolved in the same volume of buffer as the filtrate.

same volume of buffer and aliquots injected into assay mice, the NIH-ERF residue produced ESF activity equivalent to 25.99 + 0.27% 59Fe incorporation/ml. When 0.25 IU per mouse of the described NIH-ERF residue was injected ip into assay mice at various pH values with the described buffers, the optimum ESF activity response was at pH 7.4 (0.25 + 0.02 IU), and the response was minimal at pH 3.4 (0.09 + 0.01 III). The NIH-ERF activity was potentiated from 0.12 + 0 to 0.20 -+ 0.01 IU of ESF with 0.1 ml of normal rabbit serum. When 0.05 mg of EGF per mouse was dissolved in phosphate-buffered saline (pH 7.4,0.5 M NaCl) and iniected ip into eight C&I/He polycythemic assay mice, 0.39 IU (0.25-0.60) (95% limits) of erythropoietin acuvtty was produced (Table 2). When 0.2 ml of anti-EGF was incubated with 0.05 mg of EGF prior to injection, 0.12 IU (0.09-0.16) and 0.07 IU (0.04-O. 11) of erythropoietin activity was produced (16 mice). When 0.05 mg of EGF was incubated with 0.2 mg of anti-EGF, followed by centritkgation and injection of aliquots of the supernatant fluid into 16 assay mice, 0.08 IU (0.040.15) and 0.06 IU (0.04-0.09) of erythropoietin activity were produced.

404

LEWIS ET AL

TABLE 2 THE INFLUENCE OF A NEUTRALIZING ANTISERUM TO AN ERUTHROPOIE?IN-GENERATING FACTOR (EGF) ON ERYTHROPOIESIS REGULATORY FACTORS _-.-____ Percentage of 5yFe incorporated in RBC (2 SEM) --.. ..~~~-.-

ESF generated (IU (95% limits))

Experiment number

Material injected and quantity per C3HIHe mouse

530 (1) (2)

0.03 mg EGF + PBSU 0.03 mg EGF + 0.1 ml serum (NRS)

2.24 z 0.64 4.65 I 1.48

0. IX (0.07-0.30) 0.29 (0.19-0.44)

531 (1) (3 (3)” (4)’ (5)’

0.05 mg EGF + PBS 0.05 mg EGF + 0.2 ml Anti-EGF 0.05 mg EGF + 0.2 ml Anti-EGF Standard ESF, 0.16 IU Standard ESF, 0.32 IU

7.70 I.38 0.90 2.30 .5 x4

f 1.91 z 0.18 -c 0.03 z 0.69 t -‘.26

0.39 0. I? 0.08 0.14 0.21

532 (1)’

Standard ESF, 0.08 1U Standard ESF, 0.32 IU 0.05 mg EGF + 0.2 ml anti-EGF 0.05 mg EGF + 0.2 ml anti-EGF 0.16 IU ESF + 0.2 ml anti-EGF 0.16 IU ESF + 0.2 ml anti-EGF

I.29 6.5X 0.83 0.69 2.75 3.70

2 0.32 t 1.50 -c 0.14 +- 0.14 -t 1.07 f 1.29

0.09 (0.06-0.15) 0.30 (0.19-0.48) 0.07 (0.0&O. 1I) 0.06 (0.04-0.09) 0.12 (O.O&0.341 0.14 (0.05-0.36) -____

(2)’ (3) (4)” (5)’ (6)“,’

(0.25-0.60) (0.09-O. 16) (0.044 151 10.05-0.36) (0.04-1.03~

a PBS, phosphate-buffered saline; NRS, normal rabbit serum. The ECF wa:, prepared ar the VA Hospital in Augusta: the anti-EGF antiserum was produced at the Medical College of Georgia”‘,‘“‘, and the above experiments were done at the Memorial Research Center. University of Tennessee. * Solutions were centrifuged. and the supernatants were injected. p NIH-ESF.

When 0.16 IU per mouse of NIH-ESF (Table 2) was incubated with 0.2 ml of anti-EGF, the ESF activity was 0.12 IU (0.04-0.34) (eight mice). When 0.16 IU per mouse of the same ESF was incubated with 0.2 ml of anti-EGF, followed by centrifugation and injection of aliquots of the supernatant fluid into the assay mice, the ESF activity was 0.14 IU (0.05-0.36). The data obtained after incubating non-neutralizing and neutralizing anti-ESF, antiserum with ESF, are in Table 3. The data obtained after incubating neutralizing anti-ESF, antiserum with EGF are in Table 4. During polyacrylamide-gel electrophoresis of the EGF fraction, the most prominent component had a molecular weight of about 45,000. There were indications of small amounts of several components with molecular weights ranging from 69,000 to 93,000. On the most prominent component ther$ was a small shoulder with a molecular weight of 40,000, and there appeared to be a small amount of a component at the anode end of the gel with a molecular weight of 32,000 (Fig. 1). EGF could not be demonstrated in the isolated fractions.

ERYTHROPOIESIS

REGULATORY TABLE

THE

INFLUENCE

OF NON-NEUTRALIZING TO ERYTHROPOIETIN

Experiment number

3 AND

(ESF,)

405

FACTORS

ON

Material injected and quantity per CD-l mouse” Saline 0.5 mg ESF, fraction 0.5 mg ESF, fraction + 0.1 ml non-neutralizing anti-ESF, antiserum 0.5 mg ESF, fraction + 0.2 ml non-neutralizing anti-ESF, antiserum 0.5 mg ESF, fraction + 0.04 ml neutralizing anti-ESF, antiserum 0.5 mg ESF, fraction + 0.05 ml neutralizing anti-ESF, antiserum 0.5 mg ESF, fraction + 0.075 ml neutralizing anti-ESF, antiserum 0.5 mg ESF, fraction + 0.1 ml neutralizing anti-ESF, antiserum 0.5 mg ESF, fraction + 0.2 ml neutralizing anti-ESF, antiserum 0.5 mg ESF, fraction + 0.2 ml neutralizing anti-ESF, antiserum

NEUTRALIZING

ESF,

ANTISERA

ACTIVITY

Percentage of 59Fe incorporated in RBC (rSEM)

ESF activity (IU 2 SEM)

0.28 2 0.09 6.13 k 0.64

0.01 -t 0 0.13 + 0.01

13.66 2 2.83

0.30 k 0.06

14.25 2 2.06

0.32 2 0.04

0.23 ‘_ 0.08

0.01 * 0

0.19 k 0.04 .

0.004 L 0

0.27 k 0.10

0.01 2 0

0.25 ? 0.05

0.01 * 0

0.39 + 0.14

0.01 k 0

0.75 2 0.29

0.02 2 0.01

’ The antisera were prepared and the above experiments were done at the Medical College of Georgia.

DISCUSSION

Dukes et al. reported that the activity of the regulatory protein associated with their “EPO” activity was far below that of their standard EPO (erythropoietin) (9). In a previous report it was demonstrated that 0.05 mg of EGF produced ESF activity that was comparable to the activity of 0.20 mg of the ESF, fraction, when both were obtained under similar conditions (19). At the same time the production of ESF activity by EGF was shown to be pH dependent, whereas the ESF, activity was not. Later it was demonstrated that neutralizing antibodies were more readily produced with ESF, than EGF (15). The amount of EGF activity inactivated by DTT was 70.7% of the total ESF activity obtained with the NIH-ERF preparation (Table 1). The amount of EGF activity remaining in the residue on the Amicon membrane (cutoff at MW = 25,000) during filtration through centriflo cones represented 72% of the total ESF activity produced. The agreement between the expected activity of 0.38 IU with our assay of 0.34 + 0 IU for the NIH-ERF material used for the data in Table 1A is

ERYTHROPOIESIS

REGULATORY T-ABLE

~‘HE

INPLUENCE

OP NEUTRALIZING

4

AN~SERA

ON AN ESF,-GENERATING;

Experiment number I ? 3 4

5

I-O ERYTHROIWI~TIN" FACTOR

0

,

2

3

4

were

6

MOBILITY

FIG. 1. Electrophoresis ropoietin (ESF)-generating The observed molecular

6

ESF ,icticity generated IIU -e SEM

0.2x f 0.09 18.92 f 2.13

fraction per mouse fraction + 0.075 anti-ESF, antiserum fraction + 0.1 ml anti-ESF, antiserum fraction + 0.1 ml anti-ESF, antiserum fraction + 0.2 ml anti-ESF, antiserum fraction + 0.2 ml anti-ESF, antiserum fraction + 0.3 ml anti-ESF, antiserum

” Neutralizing antisera to erythropoietin done at the Medical College of Georgia.

(ESP,)

(EGF)

Percentage of “Fe incorporated in RBC (?SEM)

Material injected and quantity per CD- I mouse Saline 0.5 mg EGF 0.5 mg EGF neutralizing 0.5 mg EGF neutralizing 0.5 mg EGF neutralizing 0.5 mg EGF neutralizing 0.5 mg EGF neutralizing 0.5 mg EGF neutralizing

406

FACTORS

0.36 II 0.10 0.09 r 0.0.3 0.02

0.99

t O.‘!

o.o:! f 0.01

0.39 T 0.13 prepared

7

6

and the above

6

IO

II

12

2 0.01

11.01

experiments

2 0

were

13

km)

on SDS-polyacrylamide factor. The technique weights are as indicated.

gel of fraction containing an erythis described in Materials and Methods.

ERYTHROPOIESIS

REGULATORY

FACTORS

407

relatively good. The total value of 0.86 (Table lB), as compared to the expected value of 0.68, is significantly different. Most of the observed difference (0.18 IU) is due to the activity in the retentate, an activity believed to be comparable to the EGF production of ESF, (22). The expected value of 0.20 IU (2 x 0.10 2 0.1) for the ESF activity in the filtrate, an activity believed to be the same as ESFl, is relatively close to our assay value of 0.24 + 0.1 IU. The increased amount of erythropoiesis indicated by the total ESF activity could be explained by the removal of product inhibition. In Table 1A the ERF(s) acted in the presence of each other, whereas in Table 1B the ERF(s) had been separated. An inhibitor of erythropoiesis also could have passed into the filtrate. Increased yields of activity have been reported earlier (5). A molecular weight of 23,000 for ESF has been reported by Dorado et al. (8). We have previously used DTT to inactivate EGF, but ESF, was not inactivated (23). EGF produced about 62% of the ESF, activity in the urine from an MM patient. We reported the molecular weight of ESF, to be 20,000-30,000, and that of EGF to be 30,000-50,000 (5). The optimum pH for producing ESF activity by the NIH-ERF residues obtained from the Amicon centriflo membranes was about pH 7.4, which was also the optimum pH for producing ESF, activity with EGF both with and without normal rabbit serum (19). ESF, activity was not pH dependent; the activity was the same at pH 7.4 and 4.1. From the data obtained with anti-EGF antiserum incubated with EGF, the antiserum-neutralized EGF activity which was the equivalent of 0.29 IU of ESF before centrifugation and the equivalent of 0.32 IU after centrifugation and injection of the supernatant fluid into assay mice. In a previous report, data were presented with anti-EGF antisera which were Precipitating but non-neutralizing (15). When the anti-EGF antiserum was incubated with the NIH-ESF, there was no appreciable effect insofar as neutralization was concerned. Boosting rabbits with the NIH-ESF produced some of the highest titered antiserum yet found in that laboratory, i.e., 1 ml neutralized 75 to 100 IU of ESF (unpublished observations). Similar titers were reported elsewhere when the ESF antigen was obtained from the urine of an MM patient (24). Immunologic studies with EGF revealed that neutralizing antibodies to EGF were not readily produced, but the neutralizing immunogenic response to ESFl was relatively good (15). McDonald et al. described the production of neutralizing antibodies to the renal erythropoietic factor, erythrogenin. These antibodies also had no effect on erythronoietin (25). Neutralizing antibodies to ESF, also neutralize the ESF produced by EGF (Tables 3 and 4). Since the EGF activity could not be recovered during polyacrylamidegel electrophoresis, EGF appeared unstable under the conditions of that

408

LEWIS ET AL.

experiment. Under the same conditions erythropoietin retained stability (6,8). Previous work with the electrofractionation of ERF(s) also indicated a loss of all activity unless the ionic strength was kept high with a neutral salt (5), suggesting that a necessary component for erythropoiesis is noncovalently bound to a protein. The work with Amicon membranes and EGF isolated the EGF between membranes with molecular weight cutoffs at 50,000-30,000. The EGF exposed to polyacrylamide-gel electrophoresis was not isolated in this manner, and so those components with molecular weights appearing above 50,000 can be excluded. Additional work will be done to determine which of the remaining three proteins is the inactivated EGF. The component at 45,000 is near to the dimer size for ESF,, if the molecular weight for the ESF, monomer is accepted as 23,000 (8). As seen in Table 5, several observations are not in favor of this possibility. A variety of molecular weights have been reported for ESF (27). It has been suggested that EGF acts upon a plasma component in the TABLE OBSERVED

DIFFERENCES AND

.s

BETWEEN EGF ~~~~~~~~~~~~~~~~~ ESF, (ERYTHROPOIETIN)

Characteristic

EGF

FAC-IOR)

ESF,

Molecular weight (5) Charge above pH 7.0 (5) Dependency upon pH ( 19) Stability with dithiothreitol (23) Stability in organic solvents (26)

30,000-50.000 Cation Dependent

20.000-30.000 Anion Independent

Unstable Can be inactivated and reactivated with ethanol-acetone extract

Stable Stable

Stability during acrylamide-gel electrophoresis

Unstable

Stable (7. 8)

Type of antibodies produced

Mostly EGF-precipitating (IS) and rarely EGF-neutralizing

Mostly ESF,-neutralizing

Potentiation by normal serum (18)

Optimum potentiation at 0. I ml of normal serum; increased amounts of serum (up to 0.3 ml) indicated inhibition due to excess substrate

Initial potentiation which is unchanged by increased amounts of serum

Kinetic studies (IS. 18)

Indicated enzymatic activity

Indicated hormonal activity

ERYTHROPOIESIS

REGULATORY

FACTORS

409

assay mouse to produce ESF1, as well as during incubation with normal serum prior to injection into the assay mouse (15,19). A proper in vitro assay (one that does not require serum as a constituent or one that uses serum from which the potentiating factor has been removed) concurrent with the bioassay of the ESF activity producedlby EGF would prove or disprove this postulate. Some enzyme characteristics have been attributed to EGF, but it is not known exactly how EGF broduces ESF,. The potentiation of ESF, activity by non-neutralizing antisera to ESF, has been reported before (15,18). The kinetics of the potentiation of ESF, were different from the kinetics of the potentiation for EGF and were thought to be due to the removal of an inhibitor of erythropoiesis (EIF) by neutralizing anti-EIF antisera and/or due to the protective action of serum proteins. A number of different NIH-ERF preparations have been sent to various investigators. It would seem from the data presented that the NIH-ERF contained EGF and ESF,, whereas the NIH-ESF contained only erythropoietin. SUMMARY

A comparison

was made of two erythropoietin preparations distributed Heart and Lung Institute with two preparations from our laboratory. One NIH preparation contained a mixture of at least two erythropoiesis regulatory factors, an erythropoietin-generating factor and erythropoietin. A second NIH preparation contained erythropoietin but had no detectable generating factor.

by the National

REFERENCES 1. Gardner, E., Jr., Wright, C.-S., Lewis, J. P., and Moores. R. R. ,J. C/in. Inresr. 45, 1011 (1966) (Abstract). 2. Gardner, E., Jr., Wright, C.-S., Lewis, J. P., and Moores, R. R., Brit. J. Haemarol. 13, 317 (1967). 3. Kuratowska, Z., Lewartowski, B., and Lipinski, B., J. Lab. C/in. Med. 64, 226 (1964). 4. Gordon, A. S., Cooper, B. W., and Zanjani, E. D. Semin. Hematol. 4, 337 (1967). 5. Lewis, J. P., Neal, W. A., Welch, E. T., Lewis, W. G., III, DuBose, C. M.. Jr., Wright. C.-S., and Smith, L. L., Proc. Sot. Exp. Biol. Med. 142, 293 (1973). 6. Dorado, M., Langton, A. A., Brandon, N. C., and Espada, J., Bioc~hc~m. Med. 6, 238 (1972). 7. Espada, J., Brandon, N. C., and Dorado, M., Bioc,him. Biophys. Acta 359, 369 (1974). 8. Dorado, M., Espada, J., Langton, A. R., and Brandon, N. C., Bioc.hem. Med. 10, 1 (1974). 9. Dukes, P. P., Shore, N. A., Hammond, D., and Ortega, J. A., B/ood 43, 157 (1973) (Abstract 359). 10. Espada, J., and Gutnisky, A., Biochem. Med. 3, 475 (1970). 1I. Dukes, P. P., Hammond, D., and Shore, N. A., J. Lab. C/in. Med. 74, 250 (1969). 12. Lewis, J. P., Neal, W. A., Alford, D. A., Moores, R. R., Gardner, E., Jr., Welch, E. T., Wright, C.-S., and McWhirter, J. D., Amw. J. Vet. Ras. 31, 891 (1970).

410

ERYTHROPOIESIS

REGULATORY

FACTORS

Manual on Abnormal Hemoglobins” (7‘. H. J. Huisman. and 13. Lewis, J. P..in “Laboratory I. H. P. Jonxis, Eds.). Marcel Dekker, New York. in press. 1975. 14. Lange, R. D.. Gardner. E., Jr.. Wright, C.-S.. and Gallagher. N. I..Rut. ./. ~~c~~~~trtrro/. 10. 69 (1964). IS. Lewis. J. P.. Moores. R. R., Gardner. E.. Jr.. .Alford, D. A.. Neal. W. A.. Welch. E. T Wright. C.-S.. and Smith. L. I-.. irr “Hormones. Lipids and Misccllaneoux.” Proc. 7th Int. Congr. Clin. them., GenevaiEvian (M. Roth. J.-P. Felher. and J. .l. Scheidegger. Eds.). Vol. 3. p. 384. Karger. Basel, New York. 1969. 16. McDonald. T. P.. and Lange. R. D., .I. Ltrh. C/i,?. !&I~,~/. 70. 4X j 19671. 17. Ichiki. A. T.. and Lange. R. D.. Bir~h(,m. .%1<,