’—— if

 

 

 

BESS ELECTROPHORESIS 6F TURKEY SERUH
ALKALINE PEOSPR‘ATASE ESOENZYMES

USING. POLYACRYLAMIDE 5E1.

Thesis Foo i'm Degree of M. S.
MERE” STETE UNEWESETY
John Richard Beck
3974

 

 

‘V. 9 IFS'S

   
     

  

. LIBRARY

Michigan Sta tc
' University

 

 

ABSTRACT
DISC ELECTROPHORESIS OF TURKEY SERUM

ALKALINE PHOSPHATASE ISOENZYMES
USING POLYACRYLAMIDE GEL

By
John Richard Beck

In this study the isoenzymes of alkaline phosphatase
in turkey serum were separated by disc electrophoresis
using polyacrylamide gel as the supporting medium.
Comparisons made by previous workers between polyacrylv
amide gel and several other supporting mediums used in
electrOphoresis have generally indicated that polyacryln
amide gel will yield the greatest resolution of alkaline
phosphatase isoenzymes.

The author was unable to adapt polyacrylamide gel disc
electr0phoresis procedures that had been described by
previous workers as successful in separating the isoenzymes
of avian alkaline phosphatase for use in this laboratory.

After a period of laboratory experimentation, a rapid
and repeatable procedure that yielded excellent resolution
of alkaline phosphatase isoenzymes was devised. This pro—
cedure involved a step—by—step determination of the follow—
ing: a suitable gel system, buffer systems, staining

procedure and sample preparation.

John Richard Beck

Test subjects consisted of 103 turkeys maintained for
the "vibrator" condition at Michigan State University,
average age at first sample was 15 months :_2 months. The
females were housed in individual bird cages and the males
kept on a litter floor. A second flock of 46 female
turkeys was also tested, these birds were of the Nicholas
Broad Breasted White egg—laying strain. These birds were
housed in two-bird cages and they were 42 weeks of age at
time of sampling. Approximately one—half of the Nicholas
strain and all of the "vibrator" birds received a standard
turkey breeder ration. The remainder of the Nicholas
strain received the same ration except it contained 10
percent dried poultry anaphage. The protein content of
both rations was similar.

Blood samples were obtained from the "vibrator" flock
on two occasions: one week after the birds were subjected
to a forced molt, when egg production was zero, and at a
later date when the hens were in peak egg production. The
Nicholas strain was sampled a single time when these birds
were also in peak egg production. Egg production records
were kept in order to identify laying intensity. Semen was
collected on three occasions and individual semen production
was calculated as the average of the two most similar yields.

Thirteen distinct alkaline phosphatase bands were

isolated from the serum of the turkeys used in this study.

John Richardeeck

The thirteen different bands were observed to occur in
thirteen different patterns, each pattern being classified
as a separate zymogram. Zymogram types II, V, VI and IX
appeared only in the "vibrator" birds and zymogram types
VIII and XI appeared only in the Nicholas strain. Two
zymogram types, VI and X were observed in females only and
zymogram type XIII was observed to be limited to the males.

Zymogram type III was observed in the forceemolted
non—laying "vibrator" turkeys only and types V, VI and X
appeared only in the "vibrator" turkeys after they had been
stimulated into peak egg production. Zymogram type XII
was observed in birds classified as high intensity layers
in both the forceamolted and the peak egg production
periods. In the Nicholas strain, zymogram types I and.XI
were normally associated with the birds classified as high
intensity layers.

Isoenzyme banding patterns did not seem to be influ»
enced by either the "vibrator" condition or the difference
in the diet fed to the Nicholas strain.

The various levels of semen production did not seem
‘to be associated with any particular zymogram type.
Iiowever, when grouping together the two highest semen prOF

Cincing groups with the two highest groups of laying
intensity, zymogram types IV and V were commonly observed.

[Inder the assumption that egg—laying intensity would

John Richard Beck

represent reproductive ability in the females and semen
production would represent reproductive ability in the
males it may be possible to predict the potential reproduc—
tive ability in individuals displaying zymogram types IV

and V as opposed to the remaining zymogram types.

DISC ELECTROPHORESIS OF TURKEY SERUM
ALKALINE PHOSPHATASE ISOENZYMES

USING POLYACRYLAMIDE GEL

BY
John Richard Beck

A Thesis

Submitted to
.Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Poultry Science

1974

fAL w ACKNOWLEDGMENTS

With the completion of this thesis I realize that I have
not only learned how to conduct research or how to evaluate
and record the results, I have also profited from my associ-
ation with those people that assisted me:

Dr. T. H. Coleman and Dr. J. H. WOlford as my academic

and research advisors;

Dr. J. R. Brunner as a research advisor and committee

member;

Dr. H. C. Zindel as Chairman of the Poultry Science

Department who made available the funds and facilities

that I needed;

Mr. Sulo Hulkonen who supplied the photographs on a

short time basis;

.Mr. Mike Mangino and Mr. Larry Hood as fellow graduate

students who were invaluable in helping me solve

procedural problems.
This thesis is the embodiment of their wisdom, patience and
generosity and I am most grateful for having had the

opportunity to work with them.

TABLE

LIST OF TABLES . . . . . .

LIST OF FIGURES. . . . . .

OF CONTENTS

 

INTRODUCTION . . . . . . .
REVIEW OF LITERATURE . . .
OBJECTIVES . . . . . . . .
METHODS AND MATERIALS. . .
General . . . . . . .
Procedure . . . . . .

Apparatus . . . . . .
Chemical Formulations

Photographic Technique

PROCEDURAL EVALUATION. . .

Buffer Systems. . .

Gel System. . . . . .
Staining Procedure. .
Sample Preparation. .

RESULTS. . . . . . . . . .
DISCUSSION . . . . . . . .
SUMMARY. . . . . . . . . .
RECOMMENDATIONS. . . . . .
LITERATURE CITED . . . . .

GENERAL REFERENCES . . . .

iii

12
13

l3
16

27
29

30
30
32
33
34
37
54
60
62
63

73

TABLE

1.

LIST OF TABLES

Page

Serum alkaline phosphatase isoenzymes in the
"vibrator" turkeys at zero and peak egg
prOduCtion O O O O O O O O O O O O O I O O O 0 43

Strain differences in serum alkaline phospha—
tase isoenzymes. . . . . . . . . . . . . . . . 45

Serum alkaline phosphatase isoenzymes in the
”vibrator" turkey hens in relation to laying
intenSity. O O O O O O O O O O O O O O C O O 0 46

Serum alkaline phosphatase isoenzymes in the
Nicholas strain turkeys in relation to laying
intenSity. O O O O O O O O O O O O O O 0 O O O 48

Serum alkaline phosphatase isoenzymes in the
"vibrator" turkeys in relation to intensity
of Vibration O O O O O O O O O O O O O O O O O 49

Serum alkaline phosphatase isoenzymes in the
Nicholas strain female turkeys in relation to
diet 0 O O C O O O O O C O O O O O O O O O O O 50

Serum alkaline phosphatase isoenzymes in the
"vibrator" turkeys in relation to semen
prOduCtion O O O O O O O O O O O O O O O O O O 51

Serum alkaline phosphatase isoenzymes in
"vibrator" turkeys in relation to sex. . . . . 53

iv

LIST OF FIGURES

FIGURE Page

1. Polyacrylamide gel (disc) polymerization

raCk O O O O O O O O O O O O O O O C O O O O O 1 9

2. Polyacrylamide gel (disc) electrophoresis
cell. 0 O O O O O O O O O O O O O O O O O O O 24

3. Upper buffer reservoir, polyacrylamide gel
(disc) electrOphoresis cell . . . . . . . . . 26

4. Turkey serum alkaline phosphatase zymograms . 39

INTRODUCTION

Electr0phoresis can be loosely defined as the use of
a minute electrical current to separate protein samples
that are applied to some sort of supportative medium. In
the past a wide variety of enzymes and other proteins have
been separated using such things as: cellulose acetate
strips, paper, agar gel, starch gel and finally, polyacryl—
amide gel as the supporting medium. The advantage of using
polyacrylamide gel lies in its greater resolution capacity
for all kinds of proteins. Unlike the other forms of supv
porting.medium, polyacrylamide gel separates on the basis
of both molecular size and electrical charge.

Interaction of the polyacrylamide gel and protein
species is nonexistent. The pore size of the gel can be
varied by changing the ratio of acrylamide to bisacrylamide
and this allows the gel to act as a very effective molecular
sieve. The bisacrylamide functions as a cross linking agent
in the formation of synthetic polymers from acrylamide. The
pore size can be varied to accommodate proteins of almost
any.molecular weight.

Another important advantage of polyacrylamide gel over

other mediums is its greater ability to separate proteins

on the basis of their electrical charge. Unlike the other
supporting mediums a uniform voltage gradient is attained

in which all protein fractions of a sample will receive

an equal amount of electrical attraction from the electrodes
at all levels in the gel. Ions in the buffer function as
leading, trailing and counter ions. At the onset of cur-
rent the leading ion precedes the protein migrating anodally
(downward). The trailing ion, moving at a slower rate
anodally overtakes the protein. At the same time the
counter ion migrates cathodally (upward). The combined
movement of these three ions creates a uniform linear
voltage gradient.

Alkaline phosphatase is a group of non-specific
enzymes capable of hydrolizing a large number of primary
phOSphate esters. In vivo, these enzymes are associated
with calcification and bone formation. The obvious role
of alkaline phosphatase during egg formation has led to a
variety of investigations. Through the use of electro-
phoretic techniques the different forms of alkaline phos-
phatase (akp), termed isoenzymes, have been separated and
studied. Some of the earlier papers attempted to define
the inheritance of akp isoenzymes in several species of
fowl, however, the electrophoretic medium used was starch
gel in most cases. In a comparison of agar gel, starch
gel and polyacrylamide gel it was observed that the latter
was capable of the greatest resolution of akp isoenzymes

in chicken serum (Tamaki and Tanabe, 1970).

The onset of egg production in Japanese quail has been
observed to alter the migration rate of one isoenzyme in
polyacrylamide gel and the similar attainment of sexual
maturity in the males caused the disappearance of one
isoenzyme band. These results suggest the influence of
factors other than genetics upon the composition and elec-
trophoretic expression of akp isoenzymes. In human medicine
it has long been ascertained that diseases involving bone
and liver have profound effects on the isoenzymes as well
as total enzyme activity in serum.,

In human medicine electrophoretic assay procedures
have been clinically used in the diagnosis of a variety of
diseases. A similar procedure applicable to fowl might
possess commercial value in the selection for higher egg
production if the relation of egg formation and akp iso-

enzymes was made more explicit.

REVIEW OF LITERATURE

Alkaline phosphatase (orthophosphoric monoester phos-
phohydrolase, E.C. 3.1.3.1) is a group of non-specific
enzymes capable of hydrolyzing a large number of primary
phosphate esters regardless of the nature of the organic
radical (Posen, 1967). The term "isoenzyme" has been used
by Harris (1969) to describe different forms of an enzyme
exhibiting identical or very similar enzyme activities.

In vivo, these enzymes are associated with calcifi-
cation and bone formation (Pearse, 1968). It has been pro-
posed by Bide (1970) that the increased levels of alkaline
phosphatase (akp) in chicken plasma are the result of an
increased food consumption stimulated by increased metabolic
demand rather than being directly related to bone formation
and calcification.

In an attempt to attain a background for developing a
rapid and repeatable procedure for the separation of turkey
serum akp isoenzymes on polyacrylamide gel many papers and
books were reviewed, these papers and books are listed in

the section of this thesis entitled "General References".

Considerable expertise can be gained by reviewing these
publications.

In studying the literature it was found that a pro-
digious amount of laboratory investigation has been
accomplished with preliminary studies of either serum or
plasma akp and the separation of its isoenzymes. A number
of things have been shown to influence the activity of
akp in chicken serum and plasma. Lust and Squibb (1967),
Motzok (1950) and Sanger §E_§1. (1966) have described the
effects of certain pathological conditions on total serum
akp activity. Reports by Wilcox (1963), Bide (1970) and
Bide and Dorward (1970) have indicated the influence of
starvation upon akp activity in chicken serum. An in-
creased plasma akp in high producing hens as compared to
that of low producing hens was observed by Gutowska gt_§1.
(1943), Tanabe and Wilcox (1960) and Wilcox §E_gl. (1962).

Svozil and Pavel (1971) reported that akp activity
reached its peak in chickens just prior to egg production,
sharply diminished at onset of lay and then gradually
increased. Their data did not indicate that the akp activ—
ity recorded in layers could be correlated with these
birds' akp activity in their pre-egg laying measurement.

A correlation between enzyme activity and laying intensity
was reached only at the fourth month of egg laying. Rao

et a1. (1969) also observed maximum enzyme activity in

chickens preceding egg production. In addition, pre-lay
hens were reported to possess greater akp activity than

the same hens following one year of egg production.
Following selection for high akp activity, Wilcox et_al.
(1962) reported a heritability of .36 and estimated genetic
correlation at .50 between akp level in chicken plasma and
egg production. A phenotypic correlation of .24 was
reported by Wilcox §£_§l, (1963) between egg production and
serum akp activity in White Leghorn hens. Auchinachie and
Emslie (1934) and Stutts et;§l, (1957) were unable to corre-
late plasma akp with egg production in chickens and Common
(1936) observed no correlation between plasma akp and
intensity of lay. Using starch gel electrophoresis, Engh
and Wilcox (1971) reported that "fast" band akp frequencies
in fifteen strains of chickens selected for high egg pro-
duction ranged from .29 to .96 and thereby failed to indi-
cate any correlations between the "fast" isoenzyme and

high egg production.

Electrophoresis of Japanese quail serum on polyacryl—
amide gel by Savage gt_§l, (1970b) detected an increased
mobility of the slowest moving band upon onset of lay. In
his study the attainment of sexual maturity caused the
disappearance of one isoenzyme band.

A number of different methods of separating the iso-

enzymes of akp have been utilized with varying success.

Deactivation of isoenzymes by: heat (Moss et;al,, 1972;
Soto, 1972; It-Koon Tan §£_al., 1972), urea (Bide, 1970;
Horne §t_§l., 1969) and L-phenylalanine (Green and Antiss,
1971; Warnock, 1966; Johnson gt_§l,, 1972) have been useful
in determining tissue origin.

The stability of akp appears to be influenced by the
nature of the serum in which it is dissolved. Moss et_§l.
(1972) have observed that the pH, urea concentration and
protein concentration of the serum are minor factors influ-
encing enzyme stability.

With the use of neuraminidase-treated serum, Beckman
§E_21. (1966); Law (1967); Robinson and Pierce (1964) and
Yong (1967) demonstrated that the electrophoretic mobility
of the various isoenzymes is in a large part due to their
sialic acid content. Dymling (1966) concluded that akp
isoenzymes could not be separated on the basis of sialic
acid content or molecular weight after filtration on
Sephadex 6-200 and treatment with neuraminidase.

The removal of sialic acid residues by neuraminidase
is reported by Warnes (1972) to decrease the mobility of
human liver akp isoenzyme but has no effect on enzyme
activity.

The isoenzymes of alkaline phosphatase have been
separated by use of Sephadex G-200 chromatography (Dunne

et al., 1967; Smith et al., 1968) DEAE cellulose (anion

exchange) (Dunne gt_al., 1968) or electrophoresis using a
variety of supporting mediums.

Electrophoretic separation of akp has been conducted
on paper (Baker and Pellegrino, 1954), cellulose acetate
(Kitchener §E_§1,, 1965; Korner, 1962; Bergerman and
Blethen, 1972; Soto, 1972; Romel gt_§l., 1968; Horne gt_§1.,
1969), agar gel (Demetriou and Beattie, 1971; Maeda gt_§l.,
1972; Rawston, 1971; Dymling, 1966; Haije and De Jong,

1963; Kramer, 1968; Suzuki §E_al., 1969; Tamaki and Tanabe,
1970; Winkelman gt_§l., 1972), starch gel (Arfors §E_al.,
1963; Chiandussi gt_§1., 1962; Gahne, 1963; Green and
Antiss, 1961; Wilcox 1966; Hodson et_§l., 1962; Keiding,
1959; Langman §E_al., 1966; Robson and Harris, 1965;
Smithies, 1959; Taswell and Jeffers, 1963). Electrophoretic
separation has also been conducted on polyacrylamide gel
(Barakat §t_§1., 1971; Brown and Manley, 1970; Epstein
§E_§1., 1967; Green gt_§l,, 1972; Johnson §t_al,, 1972;
Kaplan and Rogers, 1969; Kazuga, 1972; Savage gt_al., 1970b;
Savage, 1972; Smith gt_al., 1968; Sussman and Gottlieb,
1969; Tamaki and Tanabe, 1970; Walker and Pollard, 1971).

The term "zymogram" was proposed by Hunter and Markert
(1957) to refer to cellulose strips in which the location
of enzymes was demonstrated by histochemical methods.

This term has subsequently been used in the literature to

describe any electrophoretic supporting medium that has

been specifically stained for alkaline phosphatase activity.

The fact that serum akp is subject to genetical influ-
ence has been reported in humans (Boyer, 1961; Robson and
Harris, 1965; Beckman gt_§1,, 1966), sheep (Rasmussen,
1965), cattle (Gahne, 1963), Drosophilia melanogaster
(Beckman and Johnson, 1964), bacteria (Garen, 1960),
Japanese quail (Maeda §E_§l., 1972; Savage gt_al., 1970a;
Savage, 1972), pigeons (Brown and Manley, 1970), chickens
(Law and Munro, 1965; Tamaki and Tanabe, 1970; Wilcox, 1963;
Wilcox, 1966; Stutts §E_§l., 1957) and in turkeys (Stevens
and Garza, 1968).

The great bulk of investigations using electrophoretic
methods has involved humans. The primary interest has been
directed to the relation of human akp isoenzymes to patho-
logical conditions involving liver and bone disorders. The
following supporting mediums have been used in an effort to
find a reliable, clinically useful, diagnostic tool for
human medicine:

starch gel: Skillen et al., 1972; Rosenberg, 1958;

 

Newton, 1967; Keiding, 1959; Hodson §E_§1., 1962.
agar gel: Haije and De Jong, 1963; Yong, 1967.
cellulose acetate: Bergerman and Blethen, 1972.
polyacrylamide gel: Barakat gt_§l., 1971; Connell,

1970; Epstein gt_al., 1967; Kaplan and Rogers,

1969; Kazuga, 1972; Smith et al., 1968.

10

Comparison of the different supporting mediums has
suggested that polyacrylamide gels yield the greatest
resolution of akp isoenzymes. Japanese quail serum has
been separated by polyacrylamide gel electrophoresis into
seven areas of activity by Savage §E_al. (1970a), and into
eight bands by Savage (1972). In both studies, two to four
isoenzymes were observed per sera. Separation of quail
akp on agar gel yielded six different banding patterns
(Maeda g§_al., 1972); two or three bands per sera were
observed.

Savage §E_§l. (1971) separated chicken serum akp into
five distinct bands using polyacrylamide gel. Individual
serum samples displayed one to four isoenzymes. The
separation of chicken serum akp by starch gel electrophore-
sis has yielded only a single "fast" or "slow" isoenzyme
per sample. The advantage of polyacrylamide gel over both
starch and agar gels was reported by Tamaki and Tanabe
(1970). In their study, starch and agar gel zymograms con-
sisted of two patterns containing a single "slow" or one
"slow" and one "fast" band when plasma was used. Polyacryl-
amide gel zymograms also consisted of two patterns which
displayed either two "fast" or two "slow" bands.

The best comparisons of polyacrylamide gel with other
electrOphoretic supporting mediums in the separation of akp

has been in papers dealing with human subjects. In their

11

paper comparing starch gel with polyacrylamide gel, Green
§£_§1. (1972), reported that polyacrylamide gel would
separate the liver, bone and intestinal sources of akp
isoenzymes. Similar separation with starch gel required
heat treatment of the serum in order to differentiate the
liver and bone isoenzymes.

Smith gt_gl. (1968) also reported a better resolution
of human akp isoenzymes using polyacrylamide rather than
starch gels. In their comparison of serum and tissue
extracts they were able to separate isoenzymes correspond-
ing to kidney, liver, bone and intestinal sources using
polyacrylamide gel but did not obtain similar separation
using starch gel. In both of these papers, the polyacryl-
amide gel required a lesser velume of serum containing
fewer units of enzyme activity than did starch gel.

Johnson §t_al, (1972) utilized polyacrylamide gel to
separate liver, bone, bile, intestinal and placental iso-
enzymes and normally observed two or three isoenzymes per
sera.

By use of cellulose acetate, Soto (1972) reported that
three specific forms of alkaline phosphatase exist in
humans: intestinal, placental and non-placental non-
intestinal. Normal zymograms contained two peaks of enzyme

activity when scanned colorimetrically.

OBJECTIVES

The primary objective of this study was to develop a
reliable, repeatable assay procedure for turkey serum
alkaline phosphatase using disc electrophoresis on poly-
acrylamide gel.

In the second phase of the study an attempt was made
to associate alkaline phosphatase zymograms or banding
patterns, with reproductive ability in turkeys.

In addition, the differences in zymogram types due
to sex, breed, diet and reproductive state were investi-

gated.

12

METHODS AND MATERIALS

General

Fifteen milliliter blood samples were obtained by
braChial-venous puncture from 103 turkeys selected from
the Michigan State University flock maintained for the
"vibrator" condition (Coleman §E_al., 1960). Sampling was
performed on two occasions: one week after a forced molt
(at zero egg production) and when the birds were in peak
egg production. The birds were force molted by removing
the feed and water for two days and decreasing the day
length to eight hours of artificial light per day. The
eight hour artificial light day was maintained for two
months at which time the day length was increased to four—
teen hours per day. The peak egg production sample was
taken six weeks after the fourteen hour artificial light
day was initiated. Average age at the first sampling was
15.months :_2 months. This "vibrator" flock was composed
of 21 Broad Breasted Bronze (BBB) females, 42 Broad
Breasted White (BBW) females, 16 BBB males and 24 BBW males.

A second flock of 46 BBW turkey hens was also sampled

during peak egg production. These birds were of the

13

14

Nicholas egg-laying strain. Their age at sampling was 42
weeks.

"Vibrator" hens were held in individual cages and the
males in a litter floor pen. The Nicholas strain was
housed in two bird cages.

All "vibrator" turkeys were fed "ad libitum" a standard
turkey breeder ration consisting of 17.84% protein, with
3.06% fat and 1280 kcal. metabolizable energy per pound.
Twenty—three Nicholas birds were fed the above ration, the
remaining 24 received a ration containing 10% dried poultry
anaphage (mechanically dried poultry feces). Protein con-
tent of the latter ration was assayed at 17.79%.

Collected blood was placed in a 20 ml. glass tube,
stoppered and allowed to clot. In an effort to maintain
enzyme activity, blood samples were quickly placed in
either an ice bath or a refrigerator.

Blood sampling was consistently performed in the early
.morning, approximately two hours after the lights had come
on.

Clotted blood samples were centrifuged at 10°C for
fifteen.minutes at 2500 rpm. Serum was harvested and each
individual sample placed into three one-dram.bottles for
storage at -20°C. Throughout the experiment the blood or
serum were not subjected to room temperatures for any length

of time. All serum samples were processed by polyacrylamide

15

gel electrophoresis less than one week after they were
collected.

In a preliminary experiment, blood samples were col-
1ected in a heparinized syringe. The resultant plasma was
found to be unsuitable for use in polyacrylamide gel
electrophoresis.

Daily egg production records of all hens were kept.
The Nicholas strain hens were handled in order to determine
which birds were laying. In those cages with two laying
birds, egg production was taken as an average of the total
eggs produced during the 12 day period prior to taking the
blood sample. Egg production per individual was classified
into one of four groups: A — zero eggs, B - one to three
eggs, C - four to six eggs, D — seven or more eggs.

Semen was collected on three occasions at three day
intervals. A one ml. tuberculin syringe was used to measure
semen volume. Individual semen production was calculated
as the average of the two most similar yields. Semen pro-
duction per individual was classified into one of three
groups: A — 0 to .174 ml., B - .175 to .324 ml., C - .325
ml. or greater.

"Vibrator" turkeys were classified arbitrarily on the
basis of how much shaking an individual exhibited (Coleman
§£_§1,, 1960). Birds had been classified as: Normal,

"Vibratoerinus? (infrequent and mild shaking of head),

16

"Vibrators" (moderate shaking of head) and "Vibrator plus"

(frequent and intense shaking of head).

PrOcedure

 

The procedure used to separate serum proteins was
originally described by Davis (1964). A modification of
that procedure by Savage (1972) was used to separate alka-
line phosphatase (akp)in Japanese quail. Additional changes
were made to adapt the procedure to separate Turkey akp
isoenzymes as the result of much experimentation. The pro-
cedure and apparatus used in this laboratory have proven to
be a rapid, repeatable method for electrophoretically
separating akp isoenzymes on polyacrylamide gel. Departures
from Savage's (1972) technique have served to greatly improve
resolution and perhaps decrease some of the drudgery of
using polyacrylamide gel electrophoresis. The procedure
was as follows:

1. All working solutions were removed from the refrig—

erator and allowed to reach room temperature.

2. Glass electrOphoresis tubes were soaked in column

coat solution, shaken out and air dried overnight.
The tubes were stoppered with a rubber cap that had
also been soaked in the column coat solution.

3. Tubes were inserted into two 12—place gel poly—

Amerization racks and secured with adjustment

10.

17

screws (Figure 1). To insure that all tubes were
held perfectly vertical, the rack was levelled
using a built-in spirit level.

One batch of separation gel was prepared, mixed
with a.magnetic stirrer and added to each tube in
the first rack to a scratch mark 55 mm from the
end of the tube. A 5 ml. syringe fitted with a
6.4 cm. X 20 g. needle was used.

Tubes were gently tapped with a finger to remove
all air bubbles and insure a flat lower gel surface.
Separation gel was quickly water layered to permit
polymerization and prevent meniscus formation.

A 2.54 cm piece of beveled plastic tubing was
slipped onto a 6.2 cm.x 20 g. needle to facilitate
the water layering step. A one m1. glass syringe
lubricated with Corning stopcock grease was used.
Steps four to six were repeated for the second
polymerization rack.

Frozen serum samples were removed from a —20°C
freezer and allowed to thaw.

Separation gel was allowed 30 minutes to polymer-
ize and the water layer removed with a one*ml.
syringe. Excess water was blotted with a cotton—
tipped applicator.

Stacking gel was prepared and 0.2 ml. transferred

18

.xomu coflpmNHHofihaom Aomflpv Hom oUHEmHhHomaaom

. H 9.33m

19

H musmflm

\ — -

. “It

 

ll.

12.

13.

14.

15.

16.

17.

18.

20

to each tube and the stacking gel was water
layered.

A fluorescent desk lamp was placed over both racks
at a distance of 5 cm. and photopolymerization was
allowed to proceed for 45 minutes.

Serum samples were prepared by mixing equal volumes
of serum.with 40 percent sucrose.

Tubes with completely polymerized gels were in—
8erted into the rubber grommets (after careful
removal of the rubber cap) in the upper buffer
reservoir, and then centered between the upper

and lower electrodes.

Lower reservoir was filled with 3 liters of lower
reservoir buffer. A hanging drop of buffer was
placed on the end of each separation gel to
prevent air bubble entrapment.

Upper reservoir was then placed on top of the lower
reservoir submerging the lower 3 cm. of the elec—
trophoresis tubes.

Upper reservoir was filled with 2 liters of upper
buffer and the gels were gently flushed with a
buffer filled syringe.

A 40 microliter serum sample was carefully layered
on top of the stacking gel.

A cart carrying the loaded electrophoresis cell

19.

20.

21.

22.

23.

24.

25.

26.

27.

21

and power supply was then placed in a 4°C cooler.
Electrode leads were connected, anode to the
bottom.

Power supply was switched on and the current ad-
justed to 25 milliamps for the first five minutes.
Current was then adjusted to 85 milliamps, 100
volts, for the remainder of the separation
(approximately three hours).

Turkey serum was separated until the blue colored
tracking dye had migrated to within 13 mm. of the
end of the electrophoresis tube.

Power supply was switched off and the cart returned
to the previous laboratory.

Buffer was decanted and the tubes removed from the
rubber grommets.

A 5 ml. syringe was fitted with a blunted 6.2 cm.
X 26 g. needle and was used to remove the gel by
"rimming", i.e., injecting water between the well
of tube and the gel.

Gel was then placed into a glass tube containing
15 m1. of modified incubation mixture and allowed
to react for 1.5 hours at 24°C.

Stained gels were stored in glass tubes filled with
5 percent acetic acid and allowed to destain for

48 hours before reading.

22

28. Any serum samples showing diffuse banding or light
staining were diluted 1:1 with distilled water,
centrifuged at 8500 rpm, 10°C, for fifteen minutes

and reseparated.

Apparatus

 

Electrophoretic cell (Figure 2) was constructed of
3 mm. thick plexiglas. The lower buffer reservoir was 17
cm. square and 5 cm. deep. A plexiglas tube 15 cm. long
and 5 cm. in diameter was mounted in the center of the upper
reservoir to hold the electrodes. The upper buffer reservoir
was 17 cm. square and 3 cm. deep (Figure 3). The two elec-
trodes (Figure 3-A) consisted of No. 19 platinum wire
mounted into two grooves 85 mm. apart on the plexiglas tube.
Twenty-four holes were drilled in the upper reservoir,
equally spaced and equidistant from the central electrodes.
Electrical grommets (Figure 3-B) were fitted into each hole
to hold the electrOphoresis tubes. The glass electroPhore—
sis tubes were 75.mm. in length with 5 mm. inner diameter.
The upper reservoir was fitted with L—shaped plexiglas
"legs" to facilitate loading and unloading and also to
secure it to the lower reservoir.

Power supply consisted of a Heath Regulated H.V. Power
Supply.Mode1 IP-l7. A Corning Model 12 pH meter was used

to make all pH measurements. A Sorvall RC-12 automatic

23

.Haoo mamoHOAQOHpooHo Aomflov How oUHEMHMHoomHom

.N oHsmHm

24

 

Figure 2

25

.HHmo mHmoHoomouuoon
Aomfiov How ocHEmHmHoohaom .Hwo>uommu Hommoo Home:

.m whomflm

 

26

 

3

Figure

27

refrigerated centrifuge was used to spin down all serum

samples.

Chemical Formulations

 

1. Stock Solutions (Davis, 1964; Savage, 1972)

 

A B
1 N HCl - approx. 48 m1. 1 M H3PO4 — 25.6 ml.
Tris - 36.3 gm. Tris - 5.7 gm.
TEMED - .46 m1. TEMED - .46 ml.
water to 100 m1.
titrate to pH 8.9 with HCl titrate to pH 6.9

C D
Acrylamide - 30 gm. Acrylamide — 10 gm.
Bis - .8 gm. Bis — 2.5 gm.
Water to 100 ml. water to 100 m1.

E F
Riboflavin - 4.0 gm. Sucrose - 40 gm.
water to 100 ml. water to - 60 m1.
Terms
A. Tris = 2—Amino-2-(hydroxymethyl)-l,3-propanediol

B.

C.

D.

TEMED = N,N,N',N'~Tetramethylenediamine
Bis = N,N'-Methylenebisacrylamide

Water = deionized distilled water (fresh)

Other $01uti0ns

A.

B.

Ammonium Persulfate Solution (AP)

Ammonium Persulfate - .14 gm.
Water to 100 ml.

Bromophenol Blue SOlution (BPB)

 

Bromophenol Blue - .03 gm.
Water to 100 m1.

28

Column Coat Solution

Tween 20 (Polyoxyethylene(20) Sorbitan Monolaurate
- 3 m1.

Ethylene Glycol - 3 m1.

Water - 94 m1.

Upper buffer, modified (Buchler Instruments, 1966)

Tris - 38.4 gm.
Glycine - 7.0 gm.
BPB - 2.0 m1.
Water - 2.0 liters

(titrate to pH 8.85 with Tris)

Lower Buffer (Buchler Instruments, 1966)

Tris - 29.0 gm.
11.4 N HCl- approx. 11.5 ml.
Water to 2.0 liters

(titrate to pH 8.07 with HCl)

Gel Storage Solution

 

Glacial Acetic Acid - 70 m1.

 

water - 930 m1.
'Incubation Mixture,.modified (E. C. Apparatus Co.,
St. Petersburg, Florida)
Alpha-naphthyl acid phosphate - 100 mg.
4—Amino-diphenylamine diazonium salt - 250 mg.
Magnesium sulfate - 7 H20 — 260 mg.
Maleic acid - 120 mg.
Tris - 12 gm.
water to 400 m1.

WOrking Solutions

 

Separation Gel

 

Solution A — 5 ml.
Solution C .ml.
AP — 10 m1.

I
U!

Stackinngel

 

Solution B - 1 m1.
Solution D - 2 ml.
Solution E - 1 ml.
Solution F - 3.ml.

29

Photographic Technique

Polyacrylamide gels to be photographed were placed in
20 m1. test tubes filled with gel storage solution. The
test tubes were placed on a sheet of white glass and light
was transmitted from below by means of a lS-watt fluorescent
lamp.

Exposure time was set at 1/15 (one-fifteenth) seconds
at F—8 using Panatomic-X film. Prints were made on number

three contrast paper.

PROCEDURAL EVALUATION

Upon attempting to separate the isoenzymes of turkey
akp little progress was made when using the disc electro-
phoresis procedures that were described in several of the
papers reviewed. A confusing array of buffer systems,
gel systems, sample preparations and staining procedures
have been reported. The initial experimentation in this
laboratory consisted of making:many comparisons among
those papers dealing with polyacrylamide gel electrophore-
sis in order to find an optimum procedure for separation
of turkey serum akp. As the laboratory investigation was
conducted in a series of steps the discussion will proceed

accordingly.

Buffer Systems

A suitable buffer system appears to be a crucial com-
ponent of polyacrylamide gel electrophoresis. A single
buffer can be used in both.upper and lower reservoirs
(continuous) or different buffers can be used (discontinuous).

Hjerten et'al. (1965) recommended a continuous buffer

30

31

system for optimal resolution of the protein species being
separated. In addition to the chemical constitution of

the buffer of buffers used, an alkaline pH (approximately

9 i 1 pH unit) and molar concentration ranging from .05 to
.30 are necessary for separation of alkaline phosphatase.
The maximum activity of akp was reported to occur in a .30
molar buffer (Babson gt_§1., 1966). A number of continuous
buffer systems recommended by E. C. Apparatus Co.,

St. Petersburg, Florida were investigated: Tris—Citrate
(pH 7.0), Tris—Maleic Acid (pH 9.0), Tris-Glycine (pH 8.3)
and a phosphate buffer (pH 7.0). A Tris-Borate buffer

(pH 9.5) described by Green gt;gl, (1972) was also used.
Turkey serum akp subjected to polyacrylamide gel electro-
phoresis using a continuous buffer system consistently
failed to migrate. The zymograms normally observed con—
sisted of one or two bands in close proximity to the gel
origin. In all cases of akp electrophoretic separation with
a continuous buffer system,Cyanogum-41 gel (E. C. Apparatus
Company, St. Petersburg, Florida) was used along with the
incubation mixture described in this report (Taswell and
Jeffers, 1963). The activity of akp has been reported to
be inhibited by borate (Wilcox, 1966) and Glycine (Smith
§§_§l., 1968). However, borate has been successfully used
in the electrophoretic separation of akp (Bamford et al.,
1965; Chiandussi §§_gl., 1962; Epstein et_§l., 1967; Smith

et al., 1968). Glycine was successfully incorporated into

32

the upper buffer used by Savage (1972). In the present
paper, the use of glycine in a discontinuous buffer system
yielded the best resolution of turkey akp isoenzymes.

A slight modification of the upper reservoir buffer used

by Savage (1972), resulted in a shorter separation time and
sharper bands. The modification consisted of adding addi—

tional Tris to attain a pH of 8.85.

Gel‘System

 

The initial polyacrylamide gel investigated was
Cyanogum—4l (C—41) and mixed in concentrations of 5, 7, and
10 percent. The result in all cases was diffuse banding
at the gel origin. The use of a 3 percent Cv4l "stacking"
gel had no effect on akp migration or band sharpening.

A probable cause for the poor enzyme resolution with.C—41
.may be the high percent (5.0) of cross—linking agent (Bis-
acrylamide) in the gel. The gel systems described by
Smith §E_gl. (1968), Green EELEi: (1972), and David (1964),
when used with their recommended buffers, did not yield
satisfactory results. The gel system described by Davis
(1964), was modified to produce a five percent acrylamide
gel with two and one-half percent Bisacrylamide without
success. The achievement of good isoenzyme separation was
realized in the present system in which the "separation"

gel contained 7.5% acrylamide and 2.6% bisacrylamide.

33

The importance of using a "stacking" gel has generally
been denied in the literature (Smith g£_§l., 1968; Epstein
e5;§l,, 1967; Green et_§l., 1972; Hjerten et_al., 1965).

It has been the experience in this laboratory that the
sample concentrating effect of the "stacker" gel at the
interface of the two gels will improve the resolution of

akp isoenzymes.

StainingiProcedure

Due to the non-specific nature of alkaline phosphatase
a great number of substrates have been reported in the
literature. The substrates investigated in this laboratory
included: Sodium phenolphthalein diphosphate, Sodium
phenolphthalein monophosphate, p-Nitrophenol phosphate,
Sodium beta-naphthyl acid phosphate and Sodium alpha—
naphthyl acid phosphate. The enzyme substrates were used
in combination with each of the following dye-couplers:
4-amino-diphenylamine diazonium salt, Fast Red TR salt,
Fast Blue BB salt (Gurr) and Fast Blue RR salt. The combi—
nation of Sodium alpha-naphthyl acid phosphate and 4-amino-
diphenylamine diazonium salt used in a stain buffer of pH
9.2 proved optimal (Taswell and Jeffers, 1963).

Chicken intestinal alkaline phosphatase (Sigma Chemical
Company) was used as a standard to compare histological

staining techniques. A number of different stain procedures

34

(referenced in List of References) were able to demonstrate
akp activity of this standard when dissolved in either dis-
tilled water, normal unheated serum and/or heated serum
(60°C for 15 minutes).

The staining buffers reviewed contained Magnesium
and/or Manganese for enzyme activation but the optimum pH
of the stain buffer was reported by Babson ggyal. (1966),
to be a function of substrate species and concentration.
The specific staining procedures of a number of workers
were used unsuccessfully and consequently discarded (Green
gt_§l., 1972; Maeda et;al,, 1972; Brown and Manley, 1970;
Smith gt_§1., 1968; Savage, 1972; Kaplan and Rogers, 1969;

E. C. Apparatus Co., 1971).

Sample Preparation

Throughout the literature both serum and plasma alka-
line phosphatase have been separated by electrophoresis.
As was indicated earlier, plasma was found to be unsuitable
for polyacrylamide gel electrophoresis. The absence of
enzyme migration in plasma samples could possibly be
attributed to the presence of fibrinogen. It has been
reported by Maurer (1972) that fibrinogen will migrate much
slower in particular electrophoresis gels than would be
expected due to its molecular weight. The fibrinogen mole-

cule is triangularly shaped and apparently blocks the pores

35

in polyacrylamide gel. Starch gel was reported to be un—
affected by human fibrinogen (warnock, 1966).

In this study, caution was taken to preserve enzyme
activity by cooling the blood samples and storing the serum
samples at —20°C. Gutowska gt_al. (1943) reported that the
activity of akp in plasma will decrease gradually corre-
sponding to the length of time held at room temperature.

It was recommended by Sanger et_al. (1966), that chicken
serum be assayed for akp activity as soon as possible.
Taswell and Jeffers (1963) observed that human serum could
be held for long periods of time at -20°C without appreci-
able loss of enzyme activity.

Individual serum samples were divided into three ali-
quots to facilitate repeated separations of those sera
displaying unsatisfactory enzyme banding. Thawing of serum
samples and subsequent refreezing was reported by Rendel
and Stormant (1964), to decrease the akp activity in sheep
serum by fifteen to forty percent; in addition, the decompo—
sition of the isoenzymes resulted in slower migration rates
in starch gels.

No attempt was:made during this experiment to standard—
ize the amount of enzyme activity contained in each 40 pl
serum sample undergoing electrophoretic separation. In the
case of Wilcox (1966), chicken serum was diluted with

saline to produce equal enzyme activity in all the serum

36

samples being separated on starch gel. He observed that
actual banding intensity varied from very faint to very
dark in samples of similar akp activity. In addition, he
discovered that increased dilution of serum samples re-
sulted in decreased enzyme mobility in the starch gel.

The activity of human akp was reported by Fishman and
Ghosh (1967) to increase when the serum was diluted with
water. In some cases an eighty percent increase was
observed. In the present paper the isoenzyme banding pat-
terns of pure serum and diluted serum were compared.

Turkey serum was diluted in ratios of 1:1, 1:2 and 1:3

with distilled water and "stacking" gel buffer (stock solu-
tion B). Dilution normally resulted in fainter banding
intensity while isoenzyme mobility did not appear to change.
As was mentioned earlier, dilution of turkey serum 1:1 with
distilled water was discovered to increase resolution of
the isoenzyme bands in those sera displaying poor resolu—
tion. It has been proposed by Hjerten §E_al. (1965),

that the dilution of serum will sharpen the zones of enzyme
activity during polyacrylamide gel electrophoresis due to

a decreased conductivity of the serum sample.

RESULTS

A total of thirteen distinct patterns (zymograms) of
turkey alkaline phosphatase were observed. Isoenzyme bands
were observed in thirteen different regions of the poly—
acrylamide gels. The isoenzyme possessing the highest
mobility was classified as band number one, as suggested by
Brewer (1970). The remaining bands were numbered in the
order of decreasing mobility with the slowest moving band
assigned number thirteen.

In Figure 4, a photograph is shown of each zymogram
type observed in this study accompanied by a schematic draw—
ing to help illustrate the faint bands. Each band is
numbered as indicated by the arrows. Zymograms were "read"
and classified according to zymogram type on two different
occasions. It was observed that individual gels could be
accurately classified with a repeatability of approximately
96.3%.

The data, presented in Table 1, shows each zymogram
type observed at zero and peak egg production expressed as
a percentage of the total number of each zymogram type

observed. Data for this table was obtained from the

37

38

.mEonoewm mmmumromorm mcwamxam Ednom mmxHDB

.v ousmflm

39

poocflocoo

 

 

 

 

 

 

v ounmflm

 

 

 

 

fill],
p..ll. pruglb
V '. Clo-
O a

9 .I .3-

m~ II.. as

 

 

 

 

 

 

 

 

 

40

 

 

 

 

 

Z;

 

 

 

 

concHUCOOIIv musmflm

 

 

 

90. o
5 I i

 

 

F

2,

 

 

pp

 

 

 

 

 

41

/»

«fil

 

9 .I

up

 

 

 

 

omscfluc00Ilv Guzman

 

 

 

 

 

 

 

 

 

 

42

 

 

1... 3.2.... a!
3:22,. .323

wmwpaocoottw onsmwh

‘. 0

 

43

Table 1. Serum alkaline phosphatase isoenzymes in the
"vibrator" turkeys at zero and peak egg produc—

 

 

 

 

 

tion.
Total Production Level
Zymogram number ‘ ‘(percentage of Zymograms)
type .observed. V , zero . .peak..
I 45 35.5 (16)1 64.5 (29)
II 9 33.3 ( 3) 66.7 ( 6)
III 11 100.0 (11) 0.0 ( 0)
Iv 27 74.0 (20) 26.0 ( 7)
V 15 0.0 ( 0) 100.0 (15)
VI 7 0.0 ( 0) 100.0 ( 7)
VII 25 88.0 (22) 12.0 ( 3)
IX 8 75.0 ( 6) 25.0 ( 2)
X 5 0.0 ( 0) 100.0 ( 5)
XII 33 63.3 (21) 36.7 (12)
XIII _16 25.0 ( 4) 75.0 (12)
(.201
1

that the percentage represents.

The number in parenthesis indicates the number of samples

 

44

"vibrator" flock. Zymogram type III was observed only in
the zero production period; whereas, zymogram types V, VI,
and X were observed only in the peak egg production period.

The data in Table 2 illustrates strain differences for
the zymogram types. "Vibrator" birds were observed to lack
zymogram types VIII and XI because these particular types
were present only in the Nicholas strain. Zymogram types
II, V, VI, and IX were not displayed by the Nicholas
strain. With one exception, zymogram type X which.was
observed only in the Broad Breasted White "vibrator" birds,
both "vibrator" strains displayed the same zymogram types.
In two particular instances, zymogram types I and.XII,
which involved fairly high numbers of individuals, each
"vibrator" strain possessed a reasonably equal percentage
of either type.

The data in Table 3 compares the zero production period
zymogram types to the peak production period zymogram types
of the "vibrator" hens classified as to laying intensity
during the peak production period. In the zero production
period, zymogram types I, IV, IX, and XII were observed to
appear in the birds classified as high intensity layers;
whereas, in the peak egg production period zymogram types
II, IV, V, VI, IX, and XII were associated with the high
intensity layers. Zymogram types II, IV, V, VI, IX, X, and

XII were not observed to occur in birds classified as

 

45

 

 

 

Table 2. Strain differences in serum alkaline phosphatase
Isoenzymes.
Total "Vibrator" "Vibrator" Nicholas
Zymogram number female & male female & male female
type observed BBwl BBB2 BBW
I 47 51.1 (24)3 44.6 (21) 4.2 (2)
II 9 22.2 ( 2) 77.8 ( 7) -
III 14 50.0 ( 7) 28.6 ( 4) 21.4 (3)
IV 31 58.1 (18) 29.0 ( 9) 12.9 (4)
V 15 60.0 ( 9) 40.0 ( 6) -
VI 7 71.4 ( 5) 28.6 ( 2) -
VII 34 55.9 (19) 17.6 ( 6) 26.4 (9)
VIII 3 - — 100.0 (3)
IX 8 87.5 ( 7) 12.5 ( l) -
X 10 50.0 ( 5) - 50.0 (5)
XI 8 - - 100.0 (8)
XII 42 45.2 (19) 33.3 (14) 21.4 (9)
XIII 19 68.4 (13) 15.8 ( 3) 15.8 (3)

 

lBroad Breasted White
Broad Breasted Bronze

The number in parenthesis indicates the number of indi-
vidual samples that the percentage represents.

 

46

.mucmmmumou omnucoouom

one were mmamfiom HMDUH>HocH mo Hones: mop mounoaoofl manoeudmumm cw Hones: cram
.msmw NH an

mmmo ones no r u a macho «name NH ca name ole n U @5090 «name NH ca ammo MIA
u m macho «mmmo NH Ga ammo o u e ozone “mmfluommumo mcHBOHHom one no moo ooze
oocmflmmm moz when some can moaned moo NH o How pwcHEHmumo mm3 anamooucw mswmmq

 

 

 

 

 

H
mm ma m N mm on b N om>ummoo
mamumofimu
Hones: Hopes
AS EH 3; m. “3 RH I E m. A3 54 ad m. I ”fix
any m.~ 34 RH I I I I I I x
AHV m. I I I Amy m.m Amy h.H I I NH
3 m. I I I Go o.m A3 m.m I E m. HH>
Amy ~.w Amy n.H I I I I I I H>
Amy m.m Amv o.m I I I I I I >
Amy N.v Adv m. AHV m. I AHHVN.m “av m.m Ame m.N I >H
I I I I So o.m I A: m. A: m. HHH
AS >4 A8 m.~ I I E m. I I I HH
“oav m.m Aev m.m Amy m.~ “NV h.H va m.m Amy b.m NANV >.H I H
O U m 4 Q U m d mam“
powwow cofluosooum mmm room oOHme composooum mam OHmN Emumofiha

 

Hamfioumoewn mo wmmusooummv MuflmcmucH mcfimmqi

 

 

.muflmcmucfl mcflhma on coaumHoH
ca moor moxuou =Houmuofi>= mop ca mmESNcmomH ammumrmmorm mafiamxam Eduom .m canoe

47

non—layers (i.e., zero egg production in the selected 12—
day egg production period).

The data in Table 4 shows the zymogram types in rela—
tion to egg laying intensity of the Nicholas strain.
Zymogram types I, III, and XI were not observed in the
birds classified as non—layers. Zymogram type VIII was -—
observed only in birds classified as non—layers, while
type I was observed only in birds classified as high in— %

tensity layers.

 

Table 5 shows data for each zymogram type observed in
the "vibrator" flock after separation into groups of birds
displaying the "Normal", "Vibrator", and "Vibrator-" or
"Vibrator+" conditions. No noticeable percentage of dif—
ference was observed due to the intensity of vibration.

The data in Table 6 shows the zymogram types of the
Nicholas strain in relation to diet. Except for zymogram
type VIII, which was observed only in birds that received
the standard diet, the birds on both diets had similar
percentages for all zymogram types.

The data in Table 7 shows semen volume in relation to
zymogram type. Males classified as high semen producers
(Group C) represented 30 percent of the total population
but were not associated with any particular zymogram type.
In addition, no zymogram type appeared consistently in the

males classified as low semen producers (Group A).

48

Table 4. Serum alkaline phosphatase isoenzymes in the
Nicholas strain turkeys in relation to laying
intensity.

 

 

 

 

 

 

 

 

Zymogram Laying Intensity (percentage of Zymograms)l
type .. A . B. . . C. D
2
III. - 2.2 (1) 4.4 (2) -
IV 4.4 (2) - 2.2 (l) 2.2 (1)
VII 4.4 (2) 4.4 (2) 6.6 (3) 2.2 (1)
VIII 6.6 (3) - - -
X 6.6 (3) - 2.2 (l) 2.2 (1)
x1 - 4.4 (2) 4.4 (2) 8.8 (4) 1'
XII 8.8 (4) 6.6 (3) 2.2 (l) 2.2 (1)
Total Number
Zymograms
Observed 16 8 V 12 .. 9
l

Laying intensity was determined for a 12 day period and
each bird was assigned into one of the following categories:

Group A = 0 eggs in 12 days

Group B = 1-3 eggs in 12 days

Group C = 4-6 eggs in 12 days

Group D = 7 or more eggs in 12 days

2 . . . . . .
The number 1n the parentheSIS Indicates the number of Indi-

vidual samples that the percentage represents.

49

Table 5. Serum alkaline phosphatase isoenzymes in the
"vibrator" turkeys in relation to intensity of
vibration.

 

 

‘Vibration Intensity (percentage of ZymOgrams)l

 

 

 

 

Zymogram Vibrator &
type Normal Vibrator— Vibrator+ PT
I 6.5 (13)2 9.5 (19) 6.5 (13) ‘
II 1.5 (3) 1.0 (2) 2.0 (4)
III 2.0 (4) 2.5 (5) 1.0 (2) .
IV 5.0 (10) 4.5 (9) 4.0 (8) £1
V 4.0 (8) 1.0 (2) 2.5 (5)
VI .5 (1) 1.0 (2) 2.0 (4)
VII 5.0 (10) 4.4 (9) 3.0 (6)
IX 2.0 (4) 1.5 (3) .5 (l)
X 1.0 (2) .5 (l) 1.0 (2)
XII 6.0 (12) 8.5 (17) 2.0 (2)
XIII 2.5 (5) 4.4 (9) 1.0 (2)
Total Number
Zymograms
Observed . 71 79 . . 51

 

lVibration Intensity: Normal=No shaking
Vibrator & Vibrator- = Infrequent and
mild to moderate shaking
Vibrator+ = Intense shaking

2The number in parenthesis indicates the number of individual
samples that the percentage represents.

50

Table 6. Serum alkaline phosphatase isoenzymes in the
Nicholas strain female turkeys in relation to

 

 

 

 

 

 

 

diet.
Zymogram Diet (percentage of Zymograms)
type . Standard .. I 7.10%.Poultry anaphage , “m
I 50.0 (1)1 50.0 (1)
III 33.3 (1) 66.7 (2)
IV 25.0 (1) 75.0 (3) I I
VII 66.7 (6) 33.3 (3)
VIII 100.0 (3) 0.0 (0)
x 60.0 (3) i 40.0 (2)
XI 37.5 (3) 62.5 (5)
XII 22.2 (2) 77.8 (7)
XIII 66.7 (2) 33.3 (1)
Total Number
Zymograms
Observed .. 22 .24
l

The number in parenthesis indicates the number of indi—
vidual samples that the percentage represents.

51

 

.mocmmoummu mmmucmoumm

men “may mmHoEmm Hmsofi>aocfl mo Hones: may moumofioca manorucoumm ca Hogans cram
.mooao> 30H >Hu> ou OHoN Eoum common moanom cofluopooum mom OHmN mcfluso oo>nmmoo
noesHo> cofiom .ucflom mononomon or» mm m canoe ad oowuom mafia mamfiom on» on mummomm
.HE whoa Ho mmm. u U msouw «.HE vmm.lmha. u m msouw «.HE vha.looo. u m macho ”mos
oEoHo> or» one oofinmm cofluooooum mom room oru ca pocHEHouoo wok coaposooum omfimma

 

 

 

 

 

Ame m.ma Ame m.ma HHS b.m AHV h.~ Adv h.m «av h.m HHHN
Ame N.m Amy N.m “My N.m any N.m “my ~.ma “we m.oa HHN
I I I I 3 RN 3 TN. on
I AHV h.m AHV n.m Awe N.m Amy m.ma ”HO h.~ HH>
A: BUN Amy N.m I I I I >
I I I id UN is e.m I .5
I I I So Tm is e.m I HHH
I A: Em A: UN is e.m I So Rm HH
Amy m.m Ave m.oH Ame v.m I made h.m I H
U m , m U m d
NooHHom cofluosooum mom room Nooflnmm soauosoonm mum OHmN memo
EmnmoemN

Hamfioumoemm mo omensoonwmv coauonponm :oEom

 

 

.ooauoooonm cofimm on defiumamu
cw mmoxusu enouoHoH>= one ca moexucmomfl ommooemmoom ocwamxam Enumm .h magma

52

The data in Table 8 shows the zymogram types of the
"vibrator" flock in relation to sex. Zymogram types VI
and X were observed in females only as these particular
types were absent in the males. Zymogram type XIII

appeared exclusively in male "vibrator" birds.

 

53

 

 

 

 

 

 

 

Table 8. Serum alkaline phosphatase isoenzymes in
"vibrator" turkeys in relation to sex.
Zymogram Percentage of zymogram Type
type Females Males
I 73.9 (34)1 26.1 (12)
II 66.7 (6) 27.3 (3)
III 72.7 (8) 27.3 (3)
IV 88.9 (24) 11.1 (3)
v 60.0 (9) 40.0 (6) :-
VI 100.0 (7) -
VII 52.0 (13) 48.0 (12)
IX 75.0 (6) 25.0 (2)
x 100.0 (5) -
XII 31.3 (10) 68.7 (22)
XIII - 100.0 (16)
1

The number in the parenthesis indicates the number of

individual samples that the percentage represents.

DISCUSSION

Electrophoretic separation of turkey serum akp iso-
enzymes on starch gel has previously yielded a total of
four bands (Stevens and Garza, 1968), with individual
samples displaying up to three bands. In this study elec-
trophoretic separation of turkey serum akp on polyacryl-
amide gel has revealed thirteen different bands of activity
with individual samples possessing three to six bands.

This increase in isoenzyme numbers is attributed to the use
of a more refined electrophoretic technique. Due to the
electrophoretic procedure used in this study, the actual
composition of each individual band observed in the poly-
acrylamide gels can not be determined. The existence of
thirteen distinct molecular species of alkaline phosphatase
possessing similar enzyme activity was demonstrated in this
study. Alkaline phosphatase may be similar to other enzymes
that are known to exist as isoenzymes in that the bands
observed may represent either complete polymers or their
component.monomers or dimers.

The effects of egg production in chickens and quail

upon either akp isoenzymes or total akp activity has not

54

 

55

been clarified in the literature. It has been postulated
by previous workers that chicken and quail serum akp is
controlled by several genetic and physiological factors
(Auchanachie and Emslie, 1934; Savage'et_al., 1971; Savage
e£_§l., 1970a; Savage et_§l., 1970b; Tanabe and Wilcox,
1960; Tamaki and Tanabe, 1970; RakO'et_§l,, 1964; and r‘
Stutts 3331., 1957) . ‘
Rako e§_§l. (1964) and Rao et_§l. (1969) observed
higher levels of akp in prelay hens and reduced levels when

the birds were in full egg production. Tanabe (1962) ‘

 

observed that onset of lay would increase serum akp activity.
Savage et_al. (1970a) reported an increased mobility of
quail serum isoenzyme "AKP-QO" upon onset of lay. Female
quail in egg production were observed by Savage et_§1.
(1970b) to have only "Type II" zymograms while nonlayers

and males had only "Type I" zymograms.

In an attempt to find zymograms indicative of laying
status the "vibrator" flock was sampled on two occasions;
when egg production was zero and during peak egg production.
Zymogram type III was only found in the forceemolt period
(zero egg production) of individuals of either sex. Zymo-
gram type V was observed only in the peak egg production
period of individuals of either sex. Since zymograms type
III and V were found in either sex they may reflect a

particular metabolic state rather than the effect of egg

56

production. Zymogram type VI and X were found in "high
intensity" laying females only and may indicate the influ—
ence of both egg production and.metabolism. Rako et_§1,
(1969) observed a negative correlation of akp activity with
total egg production which seemingly indicates a definite
role of akp in egg production. However, Auchanachie and pan
Emslie (1934) reported that the productivity of chickens I
was not related to plasma akp activity and that the enzyme ;

plays only a minor role in egg production.

 

The possible effect of laying intensity upon akp
activity has also been explored. Gutowska'egyal. (1943)
observed differences in total enzyme activity between good
and poor producers. Common (1936) reported that no corre-
lation existed between serum akp and intensity of egg
production as measured by‘ trap-nest records for four weeks
prior to sampling. In addition, Common (1936) observed
that past and present good egg producers exhibited similar
enzyme activity.

Egg production was measured over a twelve day period
in this study in an attempt to associate zymogram types
with laying intensity. Zymogram types II, V, VI and X
were observed in "vibrator" hens laying from four to twelve
eggs during the measurement period. In contrast, only
zymogram type IX was observed in the same group of layers

in their zero egg production period. It is interesting to

57

note that zymogram types VI and X were observed only in
females with high egg laying intensity and that these
zymograms were separated by several isoenzyme bands on the
gel.media.

The influence of genetic factors upon akp activity and
isoenzymes has been recognized for some time. Stutts'§§;§1.
(1957) consistently observed different akp activity levels
in various inbred lines of chickens. In this study zymogram
types II, V, VI, and XI were only observed in "vibrator"

birds, conversely, zymogram types VIII and XI were observed

 

 

only in the Nicholas strain. Engh and Wilcox (1971) noted
varying frequencies for a fast band in starch gel electro—
phoresis among fifteen strains of chickens, however, since
all of these strains had been selected for high.egg pro—
duction it would appear that this band was unrelated to egg
production. Savage et_§l. (1970a) suggested that isoenv
zymes AkP 89 and AkP 90 in Japanese quail were undergenetic
control. Stevens and Garza (1968) reported that bands 2 and
3 were mutually exclusive in turkeys and that codominance
could not exist between these isoenzymes. Studies of the
inheritance of chicken serum akp isoenzymes observed using
starch gel electrophoresis by Wilcox (1963, 1966) and Law
and Munro (1965) has led to the conclusion that serum akp

is controlled by a single autosomal loci exhibiting complete

dominance. The increase in total number of akp isoenzymes

58

observed in turkey serum in this study over that number
observed by Stevens and Garza (1968) suggests an improve-
ment in akp isoenzyme electrophoresis procedures. This
refined technique now offers a better opportunity to
investigate the problem of isoenzyme inheritance in turkeys
and perhaps quail and chickens as well.

Due to several generations of interbreeding, the
"vibrator" flock is assumed to be genetically similar and
such individuals would differ only by those genes responsi-

ble for the vibrator condition. Inasmuch as.most zymogram

 

types are displayed equally among the vibrator groups such
results appear to be consistent with the literature.
The minor change in protein source in the diets of
the Nicholas strain would not be expected to affect akp
zymograms. The only exception to this is zymogram type VIII,
observed in birds fed the standard diet. This particular
zymogram type was not observed in the "vibrator” flock and
its association with a particular zymogram is possibly due
to chance because of the low number of individuals involved.
Zymogram types VI and X were restricted to females
only and zymogram type XIII was observed in males only.
Sex linkage has not been reported in the literature and such
results suggest a possible influence of sex hormones instead.
Brown and Badman (1961) demonstrated the effects of exo-

genous gonadal hormones in chicks. High levels of estrogen

59

and progesterone were observed to decrease total akp activ-
ity and these effects may serve to explain the wide vari-
ations in total enzyme activity observed in some laying hens.

Zymogram types V, VI, and X were not observed during
the zero egg production period. These particular zymograms
were observed in the peak egg production period in those p1
"vibrator" hens laying four or more eggs during the twelve
day period. Such results.may suggest the influence of

estrogen upon these zymograms; however, such an idea would

 

be refuted by zymogram type III which is displayed only
during the zero egg production period by hens that were
both high and low intensity layers.

"Vibrator".males producing high semen volumes (Group C)
were not associated with any particular zymogram type(s).
Zymogram type V was observed only during the peak egg pro-
duction period and in both Group B and Group C individuals.
Zymogram types III and IV were again observed in both Group
B and Group C individuals but only during the zero egg pro-
duction period. Production of androgen by the males was
probably not as low as the production of estrogen by the
females during the zero egg production period as some of
the males tested yielded very low volumes of semen. This
fact may possibly be attributed to the lack of a specific
zymogram or zymograms associated with high semen produc-

tion (Group C).

. SUMMARY

Turkey serum akp from the Michigan State University
"Vibrator" flock and the Nicholas Broad Breasted White egg-
1aying strain has been separated by polyacrylamide gel
electrophoresis into 13 bands of activity or isoenzymes.
Following electrophoresis these activity bands were
arranged into 13 different patterns or zymograms. The in—
crease in total number of isoenzymes observed over that
observed by previous workers is attributed to an improved
electrophoretic procedure.

Zymogram type III was observed in the "vibrator" flock
only in the zero egg production period while types V, VI,

and X were observed in the peak egg production period only.

The latter two zymogram types were observed in high intensity

layers only.
Zymogram types II, V, VI, and IX were observed only in
"vibrator" birds while types VIII and XI were observed only

in the Nicholas strain.

In the zero egg production period, zymogram types I, IV,

IX, and XIII were observed to appear in the "vibrator" birds

60

 

61

classified as high intensity layers. Zymogram types II,
IV, V, VI, IX, X, and XII were not observed in birds classi-
fied as non-layers.

Zymogram type I was observed only in high intensity
layers of the Nicholas strain while type VIII was observed
only in non—layers. Zymogram types I, III, and XI were not

observed in non-layers.

 

The "vibrator" condition did not appear to affect iso-
enzyme banding pattern.

Semen production levels were not reflected by any

 

particular zymogram types.

Incorporation of 10 percent dried poultry anaphage in
the ration did not affect akp zymograms. Several zymogram
types appeared in high intensity "vibrator" layers only and
several others in high semen producers as well. Such band-
ing patterns may reflect the influence of sex hormone
status, egg production or simply a changed metabolic rate
as the result of an increased artificial light day.

Zymogram types VI and X were observed in females only
while type.XIII appeared exclusively in male "vibrator"

birds.

RECOMMENDATIONS

Further investigation into the inheritance of particu-
lar isoenzymes would yield more substantial evidence of the
influence of genetic factors in akp.

Assuming an influence of gonadal hormones upon akp

zymograms either an assay of blood hormone levels or direct

 

feeding and/or injection of such hormones may serve to '5
explain the presence of such a great variety of banding
patterns.
Identification of tissue sources of akp isoenzymes
similar to that accomplished in human medicine may also
help to explain the great number of isoenzymes observed in
this study and may lead to a greater appreciation of the
role of the isoenzymes in certain metabolic conditions and/or

disease states.

62

LITERATURE CITED

 

LITERATURE CITED

Arfors, K. E., L. Beckman and L. G. Landrin
Further Studies on the Association Between Human Serum
Phosphatases and Blood Groups.
Acta. Genet., 13:366-368, 1963

Auchanachie, D. W. and A. R. G. Emslie
The Significance of Phosphate Estimations in the Adult
Fowl.
Biochem. J., 28:1993, 1934

Babson, A. L., S. J. Greely, C. M. Coleman and G. E. Philips
Phenolphthalein Monophosphate as a Substrate for Serum
Alkaline Phosphatase.

Clin. Chem., 12:482-490, 1966

Baker, R. W. and C. Pellegrino
The Separation and Detection of Serum Enzymes by Paper
Electrophoresis.
Scand. J. Clin. Invest., 6:94, 1954

Bamford, K. F., H. Harris, J. E. Luffman, E. B. Robson and
T. E. Cleghorn
Serum Alkaline Phosphatase and the ABO Blood Groups.
Lancet, 1:530-531, 1965

Barakat, M., M. N. Grubb, R. K. Goval and T. Hersh
Intestinal Alkaline Phosphatase in Patients with Liver
Disorders.

Clin. Res. XIV, Jan. 1971, pg. 32

Beckman, L. and F. M. Johnson
Variations in Larval Alkaline Phosphatase Controlled by
Aph Alleles in Drosophilia melanogaster.
Genetics, 4:829-835, 1964

Bergerman, J. and Blethen

Determination of Alkaline PhOSphatase Isoenzymes.
Clin. Chim. Acta., 36:389-396, 1972

63

 

64

Bide, R. W.
Plasma Alkaline Phosphatase in the Fowl: Differentia-
tion of Tissue Isoenzymes by Urea.
Advan. Automat. Anal. Technicon Int. Congr., 3:169-173,
1970

Bide, R. W. and W. J. Dorward
Plasma Alkaline Phosphatase in the Fowl: Changes With
Starvation.
Poultry Sci., 49:708-713, 1970

Boyer, S. H.
Alkaline Phosphatase in Human Sera and Placenta.
Science, 134:1002, 1961

Brewer, G. J.
Introduction to Isozyme Techniques.
Academic Press Inc., New York, N. Y., 1970

Brown, W. O. and H. G. Badman
Effects of Estradiol and Progesterone on Serum Alkaline
Phosphatase in the Fowl.
Poultry Sci., 40:819—820, 1961

Brown, R. V. and J. H. Manley Jr.
Serum Alkaline Phosphatase Inheritance in the Pigeon.
Anim. Blood Grps. Biochem. Genet., 1:43-45, 1970

Buchler Instruments Inc.
1327 16th Street, Fort Lee, N. J. 07024
InstrucEIons for the Polyanalyst, an Analytical Temperaa
ture—Regulated Disc Electrophoresis Apparatus., 1966

Chiandussi, L., S. F. Greene and S. Sherlock
Serum Alkaline Phosphatase Fractions in Hepato—Biliary
and Bone Diseases.
Clin. Sci., 22:425, 1962

Coleman, T. H., R. K. Ringer, W. J..Mathey, K. G. Rood and
C. W. Pope
Vibrator, A Recessive Sex—Linked Mutatiwn in Turkeys.
J. of Hered., 51(4):158—160, 1960

Common, R. H.
Serum Phosphatase in the Domestic Fowl.
J. Agric. Sci., 26:492, 1936

Connel, M. D.
Diagnostic Use of Serum Alkaline Phosphatase Isoenzymes
and S—Nucleotidase.
Clin. Chim. Acta., 30:235-241, 1970

 

65

Davis, B. J.
Disc Electrophoresis II: Method and Application to
Human Serum Proteins.
Ann. N. Y. Acad. Sci., 121:404, 1964

Demetriou, J. A. and J. M. Beattie
Electrophoretic Separation on Agarose Thin Film of
Isoenzymes of Alkaline Phosphatase from Human Serum
and Tissue.
Clin. Chem., 17:290, 1971

Dunne, J., J. J. Fennelly and K. McGeeney
Separation of Alkaline Phosphatase Enzymes in Human
Serum Using Gel-Filtration (Sephadex G-200) Techniques.
Cancer (Philad.) 20:71-76, 1967

Dunne, J., J. J. Fennelly and K. McGeeney
Shay Chlorma Alkaline Phosphatase.
Bioehm J., 110:12P, 1968

Dymling, J. F.
Separation of Serum and Placental Alkaline Phosphatase
by Agarose Gel ElectrOphoresis and Sephadex Chromatog-
raphy.
Scand. J. Clin. Lab. Invest., 18:129, 1966

E. C. Apparatus Corporation, St. Petersburg, Fla.
Technical Bulletin 565 and Instruction Manual

Epstein, E., P. I. WOlf, J. P. Horwitz and B. Zak
An Indogenic Reaction for Alkaline Phosphatase in Disk
Electrophoresis.
Am. J. Clin. Path., 48:530, 1967

Engh, H. A. and F. H. Wilcox
Chicken Serum Alkaline Phosphatase and Egg Production.
Poultry Sci., 50:346, 1971

Fishman, W. H. and N. K. Ghosh
Isoenzymes of Human Alkaline Phosphatase.
Advances in Clin. Chem., 10:255-370, 1967

Gahne, B.
Genetic Variations of Phosphatase in Cattle Serum.
Nature, 199:305—306, 1963

Garen, A.
Genetic Control of the Specificity of the Bacterial
Enzyme Alkaline Phosphatase.
Microbial Genetics, W. Hayes and R. C. Clowed (eds.)
Cambridge Univ. Press, London and New York, pp. 239—247,
1960

 

 

66

Green, S. and C. L. Anstiss
Automated Differential Isoenzyme Analysis II. The
Fractionation of Serum Alkaline Phosphatase into
"Liver", "Intestinal" and "Other" Components.
Enzymologia, 41:9-26, 1971

Green, S., F. Cantor, N. R. Inglis and W. H. Fishman
Normal Serum Alkaline Phosphatase Isoenzymes Examined
by Acrylamide and Starch Gel Electrophoresis and by
Isoenzyme Analysis using Organ-Specific Inhibitors.
Am. J. Clin. Path., 57:52-64, 1972

Gutowska, M. S., R. T. Parkhurst, E. M. Parrot and R. M.
Verburg
Alkaline Phosphatase and Egg Formation.
Poultry Sci., 22:195, 1943

Haije, W. G. and M. DeJong
Iso-enzymes Patterns of Serum Alkaline Phosphatase in
Agar-Gel Electrophoresis and Their Clinical Signifi-
cance.
Clin. Chim. Acta., 8:620-623, 1963

Harris, H.
Genes and Isoenzymes.
Proc. Roy. Soc. (London) Ser. B, 174:1-31, 1969

Hjerten, S., S. Jerstedt and A. Tiselius
Some Aspects of the use of "Continuous" and
"Discontinuous" Buffer Systems in Polyacrylamide Gel
Electrophoresis.
Anal. Biochem., 11:219-223, 1965

Hodson, A. W., A. L. Latner and L. Raine
Iso-enzymes of Alkaline Phosphatase.
Clin. Chim. Acta., 7:255, 1962

Horne, M., C. J. Cornish and S. Posen
Use of Urea Denaturation in the Identification of
Human Alkaline Phosphatases.
J. Lab. Clin. Med., 72:905, 1969

Hunter, R. L. and C. L. Markert

 

Histochemical Demonstration of Enzymes Separated by Zone

Electr0phoresis in Starch Gel.
Science, 125:1294, 1957

It-Koon Tan, Lee-Foon Chio and Lim Teow-Suah
Heat Stability of Human Serum Alkaline Phosphatase in
the Bone and Liver Diseases.
Clin. Chim. Acta., 41:#1:329, 1972

67

Johnson, R. B., K. Ellingboe and P. Gibbs
A Study of Various Electrophoretic and Inhibition
Techniques for Separating Serum Alkaline Phosphatase
Isoenzymes.
Clin. Chem., 18:110-115, 1972

Kaplan, M. M. and L. Rogers
Separation of Human Serum Alkaline Phosphatase by
Polyacrylamide Gel Electrophoresis.
Lancet, 1:1029-1031, 1969

Kazuga, H.
Studies on a Variant Alkaline Phosphatase in Sera of
Patients with Hepatocellular Carcinoma.
Clin. Chim. Acta., 40:67, #1, 1972

 

Keiding, N. R.
Differentiation into Three Fractions of the Serum
Alkaline Phosphatase and the Behavior of the Fractions
in Diseases of Bone and Liver. ._4
Scand. J. Clin. Lab. Invest., 11:106, 1959

 

Kitchener, P. N., F. C. Neale, S. Posen and J. Brudenell-Wbods
Alkaline Phosphatase in Maternal and Fetal Sera at Term
and During Puerperium.

Am. J. Clin. Path., 44:654, 1965

Korner, N. H.
Distribution of Alkaline Phosphatase in Serum Protein
Fractions.
J. Clin. Path., 15:195, 1962

Kramer, J. W.
Isoenzymes of Serum Alkaline Phosphatase in Cats.
M. S. Thesis, 1968, Michigan State University,
E. Lansing, Mich.

Langman,.M. J., E. Lenthold, E. B. Robson, J. Harris, J. E.
Luffman and H. Harris
Influence of Diet on the "Intestinal" Component of Serum
Alkaline Phosphatase in People of Different ABO Blood
Groups and Secretor Status.
Nature (London) 212:41, 1966

Law, G. R. J.
Alkaline Phosphatase and Leucine Amino-Peptidase Associ-
ation in Plasma of the Chicken.
Science, 156:1106, 1967

68

Law, G. R. and S. S. Munro
Inheritance of Two Alkaline Phosphatase Variants in
Fowl Plasma.
Science, 149:1518, 1965

Lust, G. and R. L. Squibb
Alkaline Phosphatase Changes in Chicken Tissue During
Newcastle Disease Virus Infection.
Applied Microbiol., 15:677, 1967

Maeda, Y., T. Hashiguchi and M. Taketomi
Genetic Studies on Serum Alkaline Phosphatase Isozyme
in the Japanese Quail.
Jap. J. of Gen., 47,#3:165-170, 1972

Maurer, H. R.
Polyacrylamide Gel Electrophoresis in Clinical Chemis—
try; Problems of Standardization and Performance.
Clin. Chim. Acta., 40:#2:359, 1972

 

Moss, D. W., M. J. Shakespeare and D. M. Thomas
Observation on the Heat—Stability of Alkaline Phos-
phatase Isoenzymes in Serum.

Clin. Chim. Acta., 40:#1:35, 1972

Motzok, I.
Studies on the Plasma Phosphatase of Normal and Rachitic
Chick. 2. Relationship Between Plasma Phosphates and
the Phosphates of Bone, Kidney, Liver and Intestinal
Mucosa.
Biochem. J., 47:193-196, 1950

Newton, M. A.
Clinical Application of Alkaline Phosphatase Electro—
phoresis.
Q. J. Med., 36:17, 1967

Pearse, A. G. E.
Histochemistry, Theoretical and Applied.
Little, Brown Co., Boston, 3rd ed., 1968

Posen, S. P.
Alkaline Phosphatase.
Ann. Intern. Med., 67:183, 1967

Rako, A., F. Dumanorsky and K. Mikulec
On the Relation Between the Laying Capacity and the
Activity of Some Enzymes, the Level of Serum Proteins
and Blood Sugars in Hens.
Poultry Sci., 43:201, 1964

69

Rao, R. G., H. C. Samanna and K. T. K. Nambiar
Serum Alkaline Phosphatase Level in the Domestic Fowl.
Indian. Vet. J., 46, #4:300-303, 1969

Rasmussen, B. A.
Inheritance of R-e-i Blood Groups and Alkaline Phos-
phatase Polymorphism in Sheep.
Genetics, 51:766-770, 1965

Rawston, J. R.
Rapid Electrophoresis of Alkaline PhOSphatase Isoenzymes.
Clin. Chim. Acta., 32:303-304, 1971

 

Rendel, J. and C. Stormont
Variants of Ovine Alkaline Serum Phosphatase and Their
ABsociation with the R-O Blood Groups.
Proc. Soc. Exptl. Biol. Med., 115:853-856, 1964

Robinson, J. C. and J. E. Pierce
Differential Action of Neuraminidase on Human Serum _ 1
Alkaline PhosPhatase.

Nature, 204:472—473, 1964

 

Robson, E. B. and H. Harris
Genetics of the Alkaline Phosphatase Polymorphism.of
the Human Placenta.
Nature, 207:1257—1259, 1965

Romel, W. C., S. J. LaMancusa and J. K. DuFrene
Detection of Serum Alkaline Phosphatase Isoenzymes with
Phenolphthalein Monophosphate following Cellulose-
Acetate Electrophoresis. Clin. Chem., 14:47, 1968

Rosenberg, I. N.
Zone Electrophoresis Studies of Serum Alkaline Phos—
phatase.
J. Clin. Invest., 38:630, 1958

Sanger, V. L., R. R. Burmester and C. C. Morrill
Serum Alkaline Phosphatase Levels in Avian Osteropetro—
sis.
Avian Diseases, 10:364-371, 1966

Savage, T. F.
Genetic Studies of Serum Alkaline Phosphatase Isozymes.
Ph.D. Thesis, 1972, University of New Hampshire,
Durham, N. H.

Savage, T. F., W} M. Collins and E. C. Smith
Serum Alkaline Phosphatase Isoenzymes in Japanese Quail.
Poultry Sci., 49:1435, 1970a

70

Savage, T. F., W. M. Collins and E. C. Smith
Onset of Egg Production and Its Relationship to
Isoenzymes of Serum Alkaline Phosphatase in Japanese
Quail.
Poultry Sci., 49:1622-1644, 1970b

Savage, T. F., W. M. Collins and E. C. Smith
Detection of Isoenzymes of Chicken Serum Alkaline
Phosphatase using Polyacrylamide Disc Electrophoresis.
Poultry Sci., 50:740-743, 1971

Sigma Chemical Co., St. Louis, Mo.
Sigma Technical Bulletin No. 104, 1963

Skillen, A. W., R. D. Fifield and G. S. Sheraidah
Serum Alkaline Phosphatase Isoenzyme Patterns in
Disease.

Clin. Chim. Acta., 40:21-25, 1972

 

Smithies, O. I4
An Improved Procedure for Starch-Gel Electrophoresis:
Further Variations in the Serum Proteins of Normal
Individuals.
Biochem. J., 71:585, 1959

Smith, I., P. J. Lightstone and J. D. Perry
Separation of Human Tissue Alkaline Phosphatase by
Electrophoresis on Acrylamide Disc Gels.
Clin. Chim. Acta., 19:499-505, 1968

Soto, A.
Alkaline Phosphatase Isoenzymes.
DADE Division Amer. Hosp. Supply Corporation, 1972

Stevens, R. W. C., and J. Garza
Alkaline Phosphatase Isoenzymes in Domestic Turkeys
Proc. XIIEh Int. Cong. Genet., 1:278, 1968

Stutts, E. C., W. E. Briles and H. O. Kinkel
Plasma Alkaline Phosphatase Activity in Mature Inbred
Chickens.
Poultry Sci., 36:269-276, 1957

Sussman, H. H. and H. J. Gottlieb
Human Placental Alkaline Phosphatase II. Molecular
and Subunit Properties of the Enzyme.
Biochim. Biophys. Acta., 194:170-179, 1969

Suzuki, H., M. Yamanaka and T. Oda
Discussion Raper: Studies on Serum Alkaline Phosphatase

Isoenzymes.
Ann. N. Y. Acad. Sci., 166:811, 1969

71

Svozil, B. and J. Pavel
Applicability of Serum Alkaline Phosphatase Activity
Testing for Evaluation of Egg Production in Rhode
Island Red Hens.
Acta. Univ. Agr. Brno. Fac. Agran. l8,#3:4l3-417
(Czech), 1971

Tamaki, Y. and Y. Tanabe
Genetic Control of Multiple Molecular Forms of the
Alkaline Phosphatase in Chicken Plasma.
Poultry Sci., 49:798-804, 1970

Tanabe, Y.
Influence of Age Upon the Ability of Thyroxine and '
Estrogen to Increase Serum Alkaline Phosphatase of the
Chicken.

Gen. Comp. Endocrinol., 2:446-452, 1962

Tanabe, Y. and F. H. Wilcox .
Effects of Age, Sex and Line on Serum Alkaline Phos- r3
phatase of the Chicken.

Proc. Soc. Exptl. Biol. Med., 103:68-70, 1960

 

Taswell, H. F. and D. M. Jeffers
Isoenzymes of Serum Alkaline Phosphatase in Hepatobil-
iary and Skeletal Disease.
Am. J. Clin. Path., 40:349-356, 1963

Walker, A. W. and A. C. Pollard
Observations on Serum Alkaline Phosphatase Electrophore-
tic Patterns on Polyacrylamide Gel with Particular
Reference to the Effects of Butanol Extraction.
Clin. Chim. Acta., 34:19-29, 1971

warnes, T. W.
Progress Report: Alkaline Phosphatase.
Gut, 13:926-937, 1972

Warnock, M. L.
Characterization of Tissue and Serum Alkaline Phospha-
tase. Clin. Chim. Acta., 14:156, 1966

Wilcox, F. H.
Genetic Control of Serum Alkaline Phosphatase in the
Chicken.
J. Exp. Zool., 152:195-204, 1963

Wilcox, F. H.
A Recessively Inherited Electrophoretic variant of
Alkaline Phosphatase in Chicken Serum.
Genetics, 53:799-805, 1966

72

Wilcox, F. H., L. D. VanVleck and W. R. Harvey
Estimates of Correlation Between Serum Alkaline
Phosphatase Level and Productive Traits.
Poultry Sci., 42:1457, 1963

Wilcox, F. H., L. D. VanVleck and C. S. Shaftner
Serum Alkaline Phosphatase and Egg Production Proc.
thh_WOrld's Poultry Congress, pp. 19-22
Sydney, Australia, 1962

Winkelman, J., S. Nadler, J. Demetriou and V. J. Pileggi
The Clinical Usefulness of Alkaline Phosphatase
Isoenzyme Determinations.

Am. J. Clin. Path., 57:625-634, 1972

Yong, J. M.
Origins of Serum Alkaline Phosphatase.
J. Clin. Path., 20:647-653, 1967

 

GENERAL REFERENCES

Beckman, L.
Association Between Human Serum Alkaline Phosphatase
and Blood Groups.
Acta. Genet., 14:286, 1964

Clark, J. T.

Simplified "Disc" Electrophoresis.
Ann. N. Y. Acad. Sci., 121:428-436, 1964

Maurer, H. R.
Disc Electrophoresis and Related Techniques of Poly-
acrylamide Gel Electrophoresis.
Walter de Gruyter, Berlin.New York, 1971

Ghosh, N. K.
Purification and Molecular Properties of Placental
and Intestinal Phosphatase.
Ann. N. Y. Acad. Sci., 166:604-640, 1969

Grunder, A. A., G. E. Dickerson, A. Robertson and E. Morin
Incidence of Marek's Disease as Related to Phenotypes
of Serum Alkaline Phosphatase.

Poultry Sci., 48:1608-1611, 1969

Kuan, S. S., W. G. Martin and H. Patrich
Alkaline Phosphatase of the Chick: Partial Character—
ization of the Tissue Isoenzymes.
Proc. Soc. Exptl. Biol. Med., 122:172-177, 1966

Latner, A. L., and A. W. Hodson
Isoenzymes of Alkaline Phosphatase.
Meeting of the Assoc. of Clin. Pathologists, London,
1961
J. Clin. Path., 15:93-94

Lyons, R. B., D. D. Weaver and J. H. Beck

Isozymes of Human Leukocyte Alkaline Phosphatase.
Ann. N. Y. Acad. Sci., 155:948, 1968

73

 

GENERAL REFERENCES

 

74

Mikulec, K., and V. Mitin
Alkaline and Acid Phosphatase Activity in the Hen Blood
Serum Related to Cannabalism.
Veterinarian l9(l):143-l44, 1970

Morton, R. K.
Purification of Alkaline Phosphatase of Animal Tissue.
Biochem. J., 57:595, 1954

Moss, D. W.
The Heterogenity of Human Alkaline Phosphatase.
in: Enzymes and Isoenzymes: Structure, Properties and
Function, SEE F.E.B.S. Meeting, 18:227-239
Academic Press, London and New York, 1970

Moss, D. W. and D. M. Thomas
Modification of the Electrophoretic Mobility of Serum
Alkaline Phosphatase by Acetylation.
Clin. Chim. Acta., 30:835, 1970

Ornstein, L.
Disc Electrophoresis I: Background and Theory.
Ann. N. Y. Acad. Sci., 121:321, 1964

Raymond, S. and L. Weintraub
Acrylamide Gel As a Supporting Medium for Zone Elec-
trophoresis.
Science, 130:711, 1959

Rendel, J., O. Aalund, R. A. Freeland and F. MBller
The Relationship Between the Alkaline Phosphatase Poly—
morphism and Blood Group 0 in Sheep.
Genetics, 50:973-986, 1964

Rotenberg, S., C. Felinska and E. Gurgul
Alkaline Phosphatase Activity in Blood and the Calcium
and Phosphorus Levels in the Bones of Chickens Reared
in Cages or on the Floor.
Roez, Nauk, Roln, Ser. B. 91(1):l41-l47 (Pol.), 1969

Smith, I. (ed.)
Chromatographic and Electrophoretic Techniques; Vol.
II. Zone Electrophoresis, Second Edition
Interscience Publishers, New York, 1968

Smith, J. K., R. H. Eaton, L. G. Witby and D. W. Mess
Large—Scale Gel Filtration in the Purification of Human
Liver and Small Intestine Alkaline Phosphatase.

Anal. Biochem., 23:84, 1968

 

75

Smith, J. L. and B. L. Goodman
Heritability and Correlation for Serum Alkaline
Phosphatase and Growth in the Chick.
Poultry Sci., 49:1728-1729, 1970

Smith, J. K. and D. W. Moss
Preparative Polyacrylamide Gel Electrophoresis in the
Study of Isoenzymes.
Biochem. J., 109:44P-45P, 1968

Stevenson, D. E.
Demonstration of Alkaline Phosphatase Activity Follow-
ing Agar Gel Electrophoresis.
Clin. Chim. Acta., 6:142—143, 1961

Stolbach, L. L.
Clinical Application of Alkaline Phosphatase Isoenzyme
Analysis.
Ann. N. Y. Acad. Sci., 166:760, 1969

Sussman, H. H., P. H. Small and E. Cotlove
Immunochemical Identification of Organ-Specific Iso-
enzymes-Human Alkaline Phosphatase.
J. Biol. Chem., 243:160, 1968

Tsou, K. C.
A New Colorimetric Method for the Detenmination of
Alkaline Phosphatase with Indoxyl Phosphate.
Anal. Biochem., 11:54-64, 1965

Walker, B. A., L. C. Eze, M. C. K. Tweedie and D. A. Price-
Evans
The Influence of ABO Blood Groups, Secretor Status and
Fat Ingestion on Serum Alkaline Phosphatase.
Clin. Chim. Acta., 35:433, 1971

Wilkinson, J. H.
Isoenzymes.
J. B. Lippincott Co., Phila., Pa.
First Edition, 1966

Wilkinson, J. H.
Phenolphthalein Monophosphate as a Substrate for Serum
Alkaline Phosphatase.
Clin. Chem., 12:701—703, 1966

WOrk, T. S. and E. WOrk (eds.)
Laboratory Techniques in Biochemistry and Molecular
Biology, Vol. I.
North-Holland/American Elsevier, 1970

76

WOlf, M., A. Dinwoodie and H. G. Morgan
Comparison of Alkaline Phosphatase Isoenzymes Activity
Using Five Standard Methods.
Clin. Chim. Acta., 24:131-134, 1969