BIOCHEMICAL

Vol. 66, No. 2, 1975

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

CHARACTERIZATION INSULIN, Y. J. Topper, National

Received

OF SUPER-ACTIVE

PROLACTIN AND PLACENTAL LACTOGEN T. Oka, B. K. Vonderhaar

and M. Wilchek*

Institute of Arthritis, Metabolism and Digestive National Institutes of Health, Bethesda, Maryland

July

Diseases, 20014.

21,1975

SUMMARY: Complexes between insulin, prolactin or placental lactogen and cyanogen bromide-activated Sepharose release hormone-like materials when treated with bovine serum albumin (BSA) (1). These materials have enhanced biological activities, and are, presumably, NlN2-disubstituted guanidines in which the hormones and BSA are the substituents. The present studies show that ammonium bicarbonate can substitute Super-activity of the for BSA in the generation of the super-active hormones. can be manifested in the absence of the released, guanidinated hormones, therefore, BSA substituent. The key derivatized amino acid residues have not yet been identified, but it appears that guanidination of lysine is either unnecessary or insufficient. Several operational considerations which are important for the demonstration of these enhanced activities are discussed.

INTRODUCTION It

has been

shown

(1,2)

Sepharose

in the

prolactin

and super-active

Sepharose

complexes

prolactin

(P) and placental

in which

activities MATERIALS

are

of bovine

It

has also lactogen

essential

operational

for

insulin

serum albumin lactogen

and BSA are the

is not several

a super-active

placental

(3).

the hormone

BSA-substituent In addition,

presence

that

been (L)

(BSA).

reported

(4)

are presumably This

super-activity

considerations

Similarly,

can be released

substituents. the

can be released

relating

that

from

insulin-

a super-active

from these

the

corresponding

forms

of insulin

N1N2-disubstituted

(I)

guanidines,

report

establishes

that

of these

hormone

derivatives.

to the demonstration

the

of these

discussed.

AND METHODS

14 Ammonium bicarbonate was purchasedfrom Fisher Scientific Compapx. C-l-D-glucose (sp. act. 8.0 mC per mMole) was obtained from New England Nuclear. C-l-a-aminoisobutyric acid (sp. act. 2.4 mC per mMole) was purchased from New England Nuclear.

* On leave

from

The Department of Biophysics, Rehovot, Israel.

The WeizmarnInstitute

of Science,

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Porcine zinc-insulin was a gift from the Eli Lilly lactogen was purchased from ICN Pharmaceuticals, Inc. from the National Institutes of Health.

Company. Human placental Bovine prolactin was a gift

In the present studies, insulin-like material (ILM), prolactin-like material (PLM) and placental lactogen-like material (LLM) were generated by treating the corresponding hormone-Sepharose complexes with ammonium bicarbonate (NH4HCO ), rather than with BSA (1,3). In a standardized procedure, conducted under s 2 erile conditions, the hormone Sepharose complexes (360-440 pg per ml) suspended in freshly prepared 0.1 M NH4HCC3 solution (0.15 ml complex per 2 ml NH4HC03) were placed in plastic culture dishes and incubated in closed (to minimize change in pH due to loss of CO ) plastic boxes at 37" for 24 hours. Then the Sepharose was removed by centrifugat 2 on (500x g for 5 min), and the supernatants were lyophilized. Amino acid analyses of the dry residues indicated that 8-10 per cent of the Sepharose-linked proteins had been solubilized under these conditions. These released materials, by analogy with the materials released in the presence of BSA (4), are probably guanidines with a hormone substituent, i.e., monosubstituted guanidines. This conclusion is supported by the observation that treatment of ornithine-Sepharose with NH4HC03 generates arginine (4). RESULTS AND DISCUSSION The data in the

in Table

presence

of NH4HC03 is

of ILM released by diaphragm the

ILM stimulates

which than

Table like

i.e.,

cells

II

shows

similarly

that

(PLM),

have

It

native

from

activity

stimulates

insulin

the

to that

oxidation

of glucose

does not

exert

this

acid

(AIB)

by insulin-

from virgin

NH4HCO3 did

insulin-Sepharose

biological

of a-aminoisobutyric

with

the

placental

released

greater

of a-lactalbumin from pregnant

from

effect.

Native

mice.

not have greater

mice

is

elicited greater

indicate

not

essential

for

its

intrinsic

interest,

It

eliminates

than

that

this

super-active

material

the corresponding

Also,

insulin

biological

released

than

by these

hormones

the

effects

activity

of these

observation for

using hormones.

hormone

culture

since

of BSA (3),

of mammary explants

unmodified

derivatives.

protein,

in the

The stimulation

hormones.

by the

certain

Moreover,

794

complexes

on the guanidinated

provides a foreign

during

produced

and prolactin-

in the presence

the native

a BSA-substituent

(LLM)

Sepharose

activity

the super-activity

the necessity of the

lactogen-like

to the materials

biological

activity

The results

generation

enhanced

in mammary explants

of NH4HC03 are similar they

its

ILM released

insulin.

material

presence

but

the accumulation

treated

untreated

in

the

of BSA (1,2).

obese mice,

epithelial

had been

that

similar

in the presence from the

insensitive

I demonstrate

operational

hormones

is

In addition advantages.

such as BSA, for the nature

hormones.

the

of commercial

to

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I STIMULATORY EFFECTS OF ILM ON INSULIN-INSENSITIVE

Incubation System

TISSUES

1)

Diaphragm

j Mammary Gland 2)

Relative CO2 Production No hormone

100

Insulin ILM

148+1

Relative AIB Accumulation

100

-

100

103+5

100

-

133C8

96k2

from male C57Blf6.J 313 mice (2-3 ' in Erlenmeyer flasks containing 2 ml ~;rl~~-~~;l~~o~~ and the indicated addition of 0.5 pg material (II&f: 6.5 pg based on amino acid analyses). was determined as described previously (2). The control were in the range of 270-360 cpm/mg wet tissue/90 min. mean %S.E. of three separate experiments.

1)

Hemidiaphragms

149*10

months of age) were incubated of Medium 199 (GIBCO), 0.5 pC insulin or ins The extent of %~~l~:tuction values (expresse as 100) Each value represents the

Mammary explants from mature virgin C3H/HeN m& e (3-4 months old) were cultured 2) aa4described previously (9). The accumulation of C-a-aminoisobutyric acid ( C-AIB) was determined as described previously (1). The concentration of insulin was 5 rig/ml, and that of ILM was 0.2 pg/ml (based on amino acid analyses). The no-hormone control values (expressed as 100) were in the range of 32-40 cpm/mg wet Each value represents the mean +S.E. of four separate determinations. tissue/3 hr.

BSA is

known

to vary

the experimental The following It

was reported

insulin-resistant

from one preparation

protocols

super-activity. cribed

previously

more reproducible

considerations previously

Appropriate Moreover, (1,3),

are

(2)

diaphragm

ILM has the same activity litter-mates.

yields

to another,

that

tissue

for

insulin must,

from

on the

therefore,

when the hormone-Sepharose are

stored

at 4" in

795

demonstrating of the

tissue

of BSA from

results.

super-activity

and adipose as native

important

the elimination

sterile

these

effects.

ILM is manifested

C57B116.I

31%

corresponding be used for complexes, isotonic

mice, tissues

detection prepared saline

on but from

that lean

of the as des-

containing

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II EFFECT OF LLM AND PLM LACTOSE SYNTHETASE I Culture : Conditions

i /

Lactose formed per mg wet tissue 30 min

j Exp; 1

Ex;;

pmoles

1 o hr

2

"1;

i----------------------------------------1 ; 6 hr 1 37 IFL 30 j IFLLM / 40 57 i------------i------------------------------~ ' 24 hr 1 ; 86 i IFL

3

per "I,

-

; I 1 4 1

-

j !

c__‘E”“--__C-_‘_“--____I__ I

24 hr IFP IFPLM

/ !

I

I

:"9

::

/

Mammary gland explants from mid-pregnant C3HfHeN mice were incubated in Medium 199 [as described previously (9)] containing insulin (I) and hydrocortisone (F) at 5 pg/ml each, and one of the following: human placental lactogen (L; 1.0 ug/ml), placental lactogen-like material (LLM; 1.5 pg/ml), bovine prolactin (P; 5.0 pg/ml) or prolactin-like material (PLM; cl.5 ug/ml). All media were sterilized by passage through Millipore filters (0.45 pm pore size). The concentrations of L and P used produce maximal effects of these hormones (3). The indicated concentrations of LLM and PLM were based on amino acid analyses. LLM was generated as described in the text. PLM was generated similarly, except that 0.005 M, rather than 0.1 M NH4HCO was used. LLM and PLM were dissolved at 37" in Medium 199 immediately before the3 onset of culture. The cultures in experiments 3 and 4 were supplemented with 1% BSA. At the time of explantation (0 hr), and after 6 or 24 hours, lactose synthetase activity, a measure of the accumulation of cr-lactalbumin, was determined as described previously (3).

0.02% NaN3, they hormones

for

retain

about

lyophilization

three

their

ability

months.

of the supernatants

complexes

with

at -20".

It

NH4HC03 retain is

clearly

desirable

their

to give

rise

Furthermore, prepared

the dry after

super-activity to assay

derivatives.

796

to the soluble

promptly

residues

treatment for

only

super-active obtained

by

of the hormone-Sepharose a few weeks when stored

the super-active

hormone

BIOCHEMICAL

Vol. 66, No. 2, 1975

As suggested hormone-Sepharose also

complexes

be recalled

that

Sepharose-linked amino

acid

residue(s) focused

involved

in the

and placental

by mammary cells insulin-resistant

mammary cells (8).

form

complexes

or unnecessary

for

generation

The results and only

all

evidence

strongly

suggests

possibilities histidine

key residue, substituted

of its

of LLM,

the

can couple

residue

lysine, that

is

that

super-activity

independent

N-terminal

amino

to activated

acids,

Sepharose

ethyl-ester

can also

couple

the super-active

hormones

would

guanidines.

6 of these effect

oxidation have

by

the same

residues than

be substituted

the

insulin Also,

lysines

hormones.

from

Sepharose

contained

the super-active

homoarginine.

modified

The

hormones,

of lysine.

to be identified.

(unpublished).

hormone

of the

released

no detectable

homo-

the native

super-active

super-active

the

of into

guanidination

of the

and histidine through

converted

has 9 residues

of these

remains

acid

by insulin-sensitive

of the guanidination

residue(s)

Amino

insulin

on AIB accumulation

however,

of homoarginine.

and contained

the

did,

that

the

largely both

(6).

activity

of the hormones

showed 0.1

derivatized are

N-acetyl-tyrosine

analyses

acid

or on glucose

had no greater appeared

retained

The critical

more than

the

activity,

Accordingly,

lactogen

it

lactogen

that

It

Therefore,

10% of the

had been completely

mice,

mice.

should

to determine

reagent

insulin

Human placental converted

acid

(5).

on AIB accumulation

lactogen

by amino

of lysine,

particularly

mice.

activity.

by NH4HC03.

0.9 residue

(7)

the derivatized

supported

virgin obese

about

to be the amino

Habeeb

in

It

biological

had no stimulatory

from the

reagent

the

residue

of insulin

as insulin

insufficient

with

insulin-insensitive

The Habeeb but

either is

This

to enhanced

and Sepharose

one lysine

from pregnant

on a-lactalbumin

This

the

activity

insulin

only

In attempts

has been considered

guanidinated

diaphragm

stimulatory

is

were

leads

by NH4HC03 from the guanidines.

represent

and Methods).

which

between

released

monosubstituted

guanidination

that

from

hormones

materials

(Materials

whose

lactogen

revealed

arginine,

released

on lysine,

to homoarginine.

lysine

these

linkage

super-active are probably

hormones

we first

analyses

the

above,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Various

or tyrosine. imidazole

N-acetyl-

nitrogen If tyrosine

isoureas,

rather

and is

the than

Vol. 66, No. 2, 1975

BIOCHEMICAL

We have demonstrated are more active effects

that

biologically

have been observed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

certain than

on three

the

derivatives

of three

corresponding

native

different

biological

activities

as AIB accumulation,

activity.

Additional

hormones,

further

study

in this

animal

mouse tissues, glucose

species

oxidation

and metabolic

polypeptide hormones.

hormones The super-

and on such diverse and a-lactalbumin responses

warrant

context.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

Oka, T. and Topper, Y. J. (1974) Proc. Nat. Acad. Sci. USA 2, 1630-1633. Oka, T. and Topper, Y. J. (1975) Science 188, 1317-1319. Vonderhaar, B. K. and Topper, Y. J. (1974) Biochem. Biophys. Res. Commun. 60, 1323-1330. Wilchek, M., Oka, T., and Topper, Y. J. (1975) Proc. Nat. Acad. Sci. USA II, 1055-1058. P. (1969) Proc. Nat. Acad. Sci. USA 63, 450-457. Cuatrecasas, Habeeb, A. F. S. A. (1959) Biochim. Biophys. Acta 24, 294-296. Evans, R. L. and Saroff, H. A. (1957) J. Biol. Chem. 228, 295-304. Li, C. H., Dixon, J. S., and Chung, D. (1971) Science 173, 56-57. Elias, J. J. (1957) Science 126, 842-843.

798