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Studies on the unspecific inhibition of streptolysin O by lipids
SHORT
COMMUNICATIONS
427
fractionation described by Folch2 and with triphosphoinositide in the countercurrent distribution system described by DITTMER AND DAWSON'. This work was supported by U. S. Public Health Service Grant AM-06008 from the Institute of Arthritis and Metabolic Diseases.
solvent
Department I!nzillersity
J. DITTMER
of Biochemistry, College of Medicine, of Kentucky, Lexington, Ky. (U.S.A.)
1 2 3 4 5 6 7
J. C. D~TTMERAND R. M. C. D~WSON, Biochcm. J., 81 (1961) 535. J. FOLCH, j.Biol. Chem., 177 (1949) 497, 505. S. E. KERR, G. A. KFOURY AND L. G. DJIBELIAN, J. Lipid Res., 5 (1964) 481. C. LONG AND D. A. STAPLES,B&hem. J., 78 (1961) 179. M. A. WELLS AND J. C. DITTMER, J. Chromatog., in the press. J. C. DITTMER AND R. L. LESTER, J. Lipid Rex., 5 (1964) 126. W. D. SKIDXUORE AND C. ENTENMAN, J. Lipid Res., 3 (1962) 471. 8 S. J. THANNHAUSER, J. FELLING AND F. J. SCHMIDT,J. Biol. Chem., 215 (1955) 211. 9 M. LEES, J. FOLCH-PI, G. H. SLOANE-STANLEY AND S. J. CARR, J. Neurochem., 4 (1959) IO L. SVENNERHOLMAND H. THORIN, J. Lipid Rcs., 3 (1962) 483. II J. S. O’BRIEN, D. L. FILLERCP AND J. F. MEAD, J. Lipid Res., 5 (1964) log.
g.
Received April zznd, 1965 B&him.
Biophys.
Acta,
106 (1965)
425-42
7
SC 53064
Studies
on the unspecific inhibition
of streptolysin
0 by lipids
The haemolytic activity of streptolysin 0 (SL) can be inhibited by specific antibodies. This “antistreptolysin reaction” depends on the existence of such a specific inhibition. On the other hand, there is also an unspecific inhibition of streptolysin 0 which simulates specific antibodies. The unspecific inhibtion of streptolysin 0 was first reported by HEWITT AND TODD’ who also suggested that cholesterol was one of the streptolysin O-inhibiting substances. In addition to cholesterol a number of other substances-particularly lecithin and other phosphatidc-has been claimed to inhibit streptolysin 0. The purpose of this work was to study the nature of the unspecific inhibition of streptolysin 0, particularly with respect to the role of phosphatides. Streptolysin 0 was used according to the directions given by the manufacturer (Behringwerke, Marburg, Germany). The extent of haemolysis caused by streptolysin 0 was photometrically assayed. The extent of the unspecific inhibition of streptolysin 0 (“inhibition value”) was determined by comparing it in dilution series with the specific inhibitory effect of standard antistreptolysin (Behringwerke, Marburg, Germany). The “inhibition value” equals the number of antistreptolysin units which had the same inhibitory effect as the unspecific inhibitor. Since the cholesterol present in erythrocyte membranes is assumed to be the natural binding site of streptolysin 0 (ref. 2)) lipid extracts from human erythrocytes were used as unspecific total
lipids
inhibitors
of erythrocytes
of streptolysin
exhibited
0. Indeed,
a strong unspecific Biochim.
it was found
inhibition
Biophys.
Acta,
that
of streptolysin
the
0
106 (1965) 427-430
SHORT
428
COMMUNICATIONS
(Table I). The total lipid was separated into three fractions by colnmn chromatography (Scheme I). In several experimental series the inhibition values of these fractions were determined. In spite of the large deviations of the results both the total lipid and fraction A showed the most effective inhibition of streptolysin 0 (Table I). However Fractions B, C, D and E showed little inhibitory effect. Comparisons of the inhibiting effects with the chemical compositions of the lipid fractions revealed that fractions which were free of cholesterol (Fraction B) proved to be ineffective in spite of their high phosphatide concentrations. On the other hand, fractions which had a low phosphatide concentration (Fraction C) showed rather weak inhibitory effects in spite of their high free cholesterol concentrations. Pure cholesterol, too, had an extremely weak inhibitory effect (Table II). However, the effectiveness of a combination of cholesterol and Fraction B (which) like cholesterol showed little effect by itself) was IOOO times greater than that of cholesterol alone (Table II). On the other hand, cholesterol esters had no effect as previously reported by HOWARD et aLz, not even when combined with Fraction B. Triglycerides were also ineffective whether in combination with Fraction B or not. The increase in efficacy of cholesterol through the addition of Fraction B is obviously due to its phosphatide content. Thus, additions of chromatographically purified egg lecithin or
IK‘HIBITION
OF STREPTOLYSIN
0 UY VARIOUS LIPID FRACTIONS OF HZTI\IANERYTHROCYTES
Since the haemolytic activity of streptolysin 0 varies greatly in values were compared within the same experimental series. The lipid fractions was expressed as percentage of that of the total to 5.106 units. I’cr cent phosphatides expressed as 7; Px 25; The inhibitory activity is expressed as percentage of that of the ~____~
Lipfd
Per cmt total cholesterol (-w/w)
Pev cent phosphatides (W/WY
Inhibztory lvean
34.9* 51.x**
36.2
10” 227
Total lipid Fraction I\ Fraction I3 Fraction C Fraction D Fraction 6
0
***
**
72.5** 4.0** 10.0**
* according to **
‘4.5 93.3
KINGSLEY
0
b.h
68.8
0.5 5.6
AND
_
Xum her o_texperznzents
acti&?, .5x:
2.3
45.5*** _ _~ ._ _~_
~~.~~___
different preparations, inhibition inhibitor)- activity of the various lipid which ranged from 2.5.10~ P assav- according to TEORELL~. total Ijpid.
1 55
15
7.4 8: 5.5
IO
4
0.2
-
.~ _
4.3
:
SCHAFFERT~.
According to ZaK et LzJ.~. The remainder of Fraction E probably
contains
glycolipids.
Total lipid of human erythrocytes (extracted with ethanol-ethers) I Acetone 4 r\cetone-soluble Fraction A (61.2”,; of total lipid) Column chromatography , (SI‘1’mc acid) 4 ) Chloroform
4 Acetone-insoluble Fraction (38.896 of total lipid)
+
Fraction
C (77.1%
of total
eluate)
+
Fraction
D (13.7%
of total
eluate)
+
Fraction
E (9.276 of total
Methanol Pyridinc Scheme
Biochim.
1. Separation
Rioph_vs.
of the total
Acla.
lipid of erythrocytes.
rob (1965) 427-430
eluate)
B
SHORT COMMUNICATIONS TABLE
429
11
THI-. INHIBITORY Examples
EFFECT
OF THE ADDITION OF FRACTION B TO VARIOUS LIPIDS
of several experimental
series. Initial lipid concentration,
I mg/ml.
_____~~
_
Lipid
Inhibition value of the substance alone
Inhibition value o,f the mixture qf the lipzd with Fraction B
Relation Lipid: Fraction R (w/v)
Cholesterol Cholesterol acetate Cholesterol stearate Tripalmitin Tristearin
60 0 o < 20 < 20
6.10~ o 0 < 20 < 20
I : 0.96 I : 0.50 I : 0.50 I : 0.66 I : 0.60
crude soya-bean phosphatides also increased the inhibitory ability of cholesterol. This auxiliary effect of phosphatides is apparently due to their functioning as solvating agents by which the dispersion of cholesterol and the number of its reacting groups --viz. $-hydroxyl group2---is greatly increased. RAPPORT’ has recently pointed to the auxiliary effect of phosphatides in immunological systems. This can be understood from the peculiar physicochemical properties of phosphatides as shown by BANGHAM~ Pure
and VAN DEENEN~~. phosphatides
by phosphatides
(e.g. ref.
do not inhibit I)
are probably
0. Reports due to contamination
streptolysin
with cholesterol or with related sterols. Further experiments concerning the mechanism will be reported later ll.
on such inhibitions of the preparations
of the action of streptolysin
Physiologisch-Chemisches Institut der Universittit Giittingen, K.F. Hygiene-Institut der Universitiit, Freiburg i.Br. and Tierhygienisches Institut, Freiburg i.Br. (Germany)
0
0. Vv’. THIELE PETERSEN
AND P. NOWAK
B. URB.4SCHEK
I L. F. HEWITT .%ND E. W. TODD, J. Pathol. Bacterial., 4g (1939) 45, 2 J. G. HOWARD, K. R. WALLACE AND A. B. WRIGHT, Brit. J. Exptl. Pathol., 34 (1953) 174. 3 B. URBASCHEK AND 0. W. THIELE, 2. Immunitiitsforsch., 126 (1964) 280. 4 B. BORGSTRGM, Acta Physiol. Stand., 25 (1952) IOI. 5 G. R. KINGSLEY AND R. SCHAFFERT, J. Biol. Chem., 180 (1949) 315. 6 B. ZAK, R. C. DICKENMAN, E. G. WHITE, W. BURNETT AND P. J. CHERNEY, Am. J. Clin. Pathol., 24 (1954) 1307. 7 T. TEORELL, Biochem. Z., 230 (1931) I. 8 M. M. RAPPORT, J. Lipid RES., 2 (1961) 25. g A. D. BANGHAM, in Advances in Lipid Research, Vol. I, Academic Press, New York, 1963. p. 65. IO L. L. M. VAN DEENEN, Physico-Chemical Aspects of Lipid Structures in Membranes. 6th Internat. Congr. Biochem., New York, 1964. I I K. I:. PETERSEN, P. NOWAK, 0. W. THIELEANI) B. URBASCHEK, Intern. Arch. Allergy,
Received
May xrst,
in thepress.
1965 B&him.
Biophys.
Ada,
106 (1965)
427-429
SHORT COMMUNIC;ZTIONS
430 SC 53066
Acetyl-CoA carboxylase and citrate cleavage enzymein the rat mammary gland Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the rate-limiting reaction in that citrate fatty acid synthesis from acetate in various systems I. The demonstration can serve as an efficient precursor for fatty acids 2,3 led SPENCER et aL4 to suggest that citrate cleavage enzyme might catalyze the rate-limiting step under physiological conditions. Direct comparison of the levels of acetate thiokinase (EC 6.2.1.1) and citrate cleavage enzyme in the livers of rats under various nutritional conditions5 supported the concept that citrate served as a more efficient precursor for extramitochondrial fatty acid synthesis than acetate, but left unanswered the relative significance of citrate cleavage enzyme and acetyl-CoA carboxylase in this process. We sought to resolve this question by comparing directly the levels of acetyl-CoA carboxylase and citrate cleavage enzyme in the rat mammary gland under conditions of the altered fatty acid synthesis accompanying lactation and its onset and cessation. The preparation of zoooo x g supernatant fractions from mammary extracts has been describedB. High-speed supernatant fractions were obtained from these by centrifuging at 105 ooo x g for I h and carefully withdrawing clear solution devoid of loosely sedimented particles or fat. Glycerol was added to the 20000 x g and 105ooo x supernatant solutions to 20% (v/v); these enzyme solutions are referred to as SpZO and SplO, respectively. Acetyl-CoA carboxylase was assayed as previously reportedB. Incorporation of H%O,was proportional to incubation time and enzyme concentration, at protein concentrations below 0.3 mg/mI. Experiments conducted with [Xlmalonyl-CoA under conditions of the acetyl-CoA carboxylase assay provided no evidence for the presence of an interfering malonyl-CoA decarboxylase, or a malonyl-CoA-CO, exchange reaction. Citrate cleavage enzyme was assayed spectrophotometrically by coupling with malate dehydrogenase ‘, (EC 1.1.1.37) which was present in great excess in all preparations. Kates of NADH oxidation were strictly proportional to protein concentration and linear with time. The milk content of each enzyme preparation was calculated from its lactose concentration6. Protein concentrations* of the enzyme solutions were corrected for milk protein by determining their lactose content, and relating this to the lactose and protein concentrations of rat milk Sp,, and Sp,,,,. Fig. I illustrates the similarity in the patterns of acetyl-CoA carboxylase and citrate cleavage enzyme activities throughout the lactation cycle, Roth enzymes rise rapidly during the first 4 days of lactation, remain at high levels and drop precipitously when the young are removed. This pattern, which parallels the alteration in fatty-acid synthesis in the mammary gland under the same conditions9suggests an important role for both enzymes in mammary fatty-acid synthesis. ,~BR.~HAM (cited in SREREIO), has demonstrated the concurrent alteration in the rate of fatty-acid synthesis and citrate cleavage enzyme activity during the lactation cycle in the rat mammary gland. Similar results were reported by LOWENSTEIN et aLlI. The specific activity of citrate cleavage enzyme is always higher than that of acetyl-Co.4 carB&him. Biophys.
Acta,
IOG (1965) 430-433
COMMUNICATIONS
427
fractionation described by Folch2 and with triphosphoinositide in the countercurrent distribution system described by DITTMER AND DAWSON'. This work was supported by U. S. Public Health Service Grant AM-06008 from the Institute of Arthritis and Metabolic Diseases.
solvent
Department I!nzillersity
J. DITTMER
of Biochemistry, College of Medicine, of Kentucky, Lexington, Ky. (U.S.A.)
1 2 3 4 5 6 7
J. C. D~TTMERAND R. M. C. D~WSON, Biochcm. J., 81 (1961) 535. J. FOLCH, j.Biol. Chem., 177 (1949) 497, 505. S. E. KERR, G. A. KFOURY AND L. G. DJIBELIAN, J. Lipid Res., 5 (1964) 481. C. LONG AND D. A. STAPLES,B&hem. J., 78 (1961) 179. M. A. WELLS AND J. C. DITTMER, J. Chromatog., in the press. J. C. DITTMER AND R. L. LESTER, J. Lipid Rex., 5 (1964) 126. W. D. SKIDXUORE AND C. ENTENMAN, J. Lipid Res., 3 (1962) 471. 8 S. J. THANNHAUSER, J. FELLING AND F. J. SCHMIDT,J. Biol. Chem., 215 (1955) 211. 9 M. LEES, J. FOLCH-PI, G. H. SLOANE-STANLEY AND S. J. CARR, J. Neurochem., 4 (1959) IO L. SVENNERHOLMAND H. THORIN, J. Lipid Rcs., 3 (1962) 483. II J. S. O’BRIEN, D. L. FILLERCP AND J. F. MEAD, J. Lipid Res., 5 (1964) log.
g.
Received April zznd, 1965 B&him.
Biophys.
Acta,
106 (1965)
425-42
7
SC 53064
Studies
on the unspecific inhibition
of streptolysin
0 by lipids
The haemolytic activity of streptolysin 0 (SL) can be inhibited by specific antibodies. This “antistreptolysin reaction” depends on the existence of such a specific inhibition. On the other hand, there is also an unspecific inhibition of streptolysin 0 which simulates specific antibodies. The unspecific inhibtion of streptolysin 0 was first reported by HEWITT AND TODD’ who also suggested that cholesterol was one of the streptolysin O-inhibiting substances. In addition to cholesterol a number of other substances-particularly lecithin and other phosphatidc-has been claimed to inhibit streptolysin 0. The purpose of this work was to study the nature of the unspecific inhibition of streptolysin 0, particularly with respect to the role of phosphatides. Streptolysin 0 was used according to the directions given by the manufacturer (Behringwerke, Marburg, Germany). The extent of haemolysis caused by streptolysin 0 was photometrically assayed. The extent of the unspecific inhibition of streptolysin 0 (“inhibition value”) was determined by comparing it in dilution series with the specific inhibitory effect of standard antistreptolysin (Behringwerke, Marburg, Germany). The “inhibition value” equals the number of antistreptolysin units which had the same inhibitory effect as the unspecific inhibitor. Since the cholesterol present in erythrocyte membranes is assumed to be the natural binding site of streptolysin 0 (ref. 2)) lipid extracts from human erythrocytes were used as unspecific total
lipids
inhibitors
of erythrocytes
of streptolysin
exhibited
0. Indeed,
a strong unspecific Biochim.
it was found
inhibition
Biophys.
Acta,
that
of streptolysin
the
0
106 (1965) 427-430
SHORT
428
COMMUNICATIONS
(Table I). The total lipid was separated into three fractions by colnmn chromatography (Scheme I). In several experimental series the inhibition values of these fractions were determined. In spite of the large deviations of the results both the total lipid and fraction A showed the most effective inhibition of streptolysin 0 (Table I). However Fractions B, C, D and E showed little inhibitory effect. Comparisons of the inhibiting effects with the chemical compositions of the lipid fractions revealed that fractions which were free of cholesterol (Fraction B) proved to be ineffective in spite of their high phosphatide concentrations. On the other hand, fractions which had a low phosphatide concentration (Fraction C) showed rather weak inhibitory effects in spite of their high free cholesterol concentrations. Pure cholesterol, too, had an extremely weak inhibitory effect (Table II). However, the effectiveness of a combination of cholesterol and Fraction B (which) like cholesterol showed little effect by itself) was IOOO times greater than that of cholesterol alone (Table II). On the other hand, cholesterol esters had no effect as previously reported by HOWARD et aLz, not even when combined with Fraction B. Triglycerides were also ineffective whether in combination with Fraction B or not. The increase in efficacy of cholesterol through the addition of Fraction B is obviously due to its phosphatide content. Thus, additions of chromatographically purified egg lecithin or
IK‘HIBITION
OF STREPTOLYSIN
0 UY VARIOUS LIPID FRACTIONS OF HZTI\IANERYTHROCYTES
Since the haemolytic activity of streptolysin 0 varies greatly in values were compared within the same experimental series. The lipid fractions was expressed as percentage of that of the total to 5.106 units. I’cr cent phosphatides expressed as 7; Px 25; The inhibitory activity is expressed as percentage of that of the ~____~
Lipfd
Per cmt total cholesterol (-w/w)
Pev cent phosphatides (W/WY
Inhibztory lvean
34.9* 51.x**
36.2
10” 227
Total lipid Fraction I\ Fraction I3 Fraction C Fraction D Fraction 6
0
***
**
72.5** 4.0** 10.0**
* according to **
‘4.5 93.3
KINGSLEY
0
b.h
68.8
0.5 5.6
AND
_
Xum her o_texperznzents
acti&?, .5x:
2.3
45.5*** _ _~ ._ _~_
~~.~~___
different preparations, inhibition inhibitor)- activity of the various lipid which ranged from 2.5.10~ P assav- according to TEORELL~. total Ijpid.
1 55
15
7.4 8: 5.5
IO
4
0.2
-
.~ _
4.3
:
SCHAFFERT~.
According to ZaK et LzJ.~. The remainder of Fraction E probably
contains
glycolipids.
Total lipid of human erythrocytes (extracted with ethanol-ethers) I Acetone 4 r\cetone-soluble Fraction A (61.2”,; of total lipid) Column chromatography , (SI‘1’mc acid) 4 ) Chloroform
4 Acetone-insoluble Fraction (38.896 of total lipid)
+
Fraction
C (77.1%
of total
eluate)
+
Fraction
D (13.7%
of total
eluate)
+
Fraction
E (9.276 of total
Methanol Pyridinc Scheme
Biochim.
1. Separation
Rioph_vs.
of the total
Acla.
lipid of erythrocytes.
rob (1965) 427-430
eluate)
B
SHORT COMMUNICATIONS TABLE
429
11
THI-. INHIBITORY Examples
EFFECT
OF THE ADDITION OF FRACTION B TO VARIOUS LIPIDS
of several experimental
series. Initial lipid concentration,
I mg/ml.
_____~~
_
Lipid
Inhibition value of the substance alone
Inhibition value o,f the mixture qf the lipzd with Fraction B
Relation Lipid: Fraction R (w/v)
Cholesterol Cholesterol acetate Cholesterol stearate Tripalmitin Tristearin
60 0 o < 20 < 20
6.10~ o 0 < 20 < 20
I : 0.96 I : 0.50 I : 0.50 I : 0.66 I : 0.60
crude soya-bean phosphatides also increased the inhibitory ability of cholesterol. This auxiliary effect of phosphatides is apparently due to their functioning as solvating agents by which the dispersion of cholesterol and the number of its reacting groups --viz. $-hydroxyl group2---is greatly increased. RAPPORT’ has recently pointed to the auxiliary effect of phosphatides in immunological systems. This can be understood from the peculiar physicochemical properties of phosphatides as shown by BANGHAM~ Pure
and VAN DEENEN~~. phosphatides
by phosphatides
(e.g. ref.
do not inhibit I)
are probably
0. Reports due to contamination
streptolysin
with cholesterol or with related sterols. Further experiments concerning the mechanism will be reported later ll.
on such inhibitions of the preparations
of the action of streptolysin
Physiologisch-Chemisches Institut der Universittit Giittingen, K.F. Hygiene-Institut der Universitiit, Freiburg i.Br. and Tierhygienisches Institut, Freiburg i.Br. (Germany)
0
0. Vv’. THIELE PETERSEN
AND P. NOWAK
B. URB.4SCHEK
I L. F. HEWITT .%ND E. W. TODD, J. Pathol. Bacterial., 4g (1939) 45, 2 J. G. HOWARD, K. R. WALLACE AND A. B. WRIGHT, Brit. J. Exptl. Pathol., 34 (1953) 174. 3 B. URBASCHEK AND 0. W. THIELE, 2. Immunitiitsforsch., 126 (1964) 280. 4 B. BORGSTRGM, Acta Physiol. Stand., 25 (1952) IOI. 5 G. R. KINGSLEY AND R. SCHAFFERT, J. Biol. Chem., 180 (1949) 315. 6 B. ZAK, R. C. DICKENMAN, E. G. WHITE, W. BURNETT AND P. J. CHERNEY, Am. J. Clin. Pathol., 24 (1954) 1307. 7 T. TEORELL, Biochem. Z., 230 (1931) I. 8 M. M. RAPPORT, J. Lipid RES., 2 (1961) 25. g A. D. BANGHAM, in Advances in Lipid Research, Vol. I, Academic Press, New York, 1963. p. 65. IO L. L. M. VAN DEENEN, Physico-Chemical Aspects of Lipid Structures in Membranes. 6th Internat. Congr. Biochem., New York, 1964. I I K. I:. PETERSEN, P. NOWAK, 0. W. THIELEANI) B. URBASCHEK, Intern. Arch. Allergy,
Received
May xrst,
in thepress.
1965 B&him.
Biophys.
Ada,
106 (1965)
427-429
SHORT COMMUNIC;ZTIONS
430 SC 53066
Acetyl-CoA carboxylase and citrate cleavage enzymein the rat mammary gland Acetyl-CoA carboxylase (EC 6.4.1.2) catalyzes the rate-limiting reaction in that citrate fatty acid synthesis from acetate in various systems I. The demonstration can serve as an efficient precursor for fatty acids 2,3 led SPENCER et aL4 to suggest that citrate cleavage enzyme might catalyze the rate-limiting step under physiological conditions. Direct comparison of the levels of acetate thiokinase (EC 6.2.1.1) and citrate cleavage enzyme in the livers of rats under various nutritional conditions5 supported the concept that citrate served as a more efficient precursor for extramitochondrial fatty acid synthesis than acetate, but left unanswered the relative significance of citrate cleavage enzyme and acetyl-CoA carboxylase in this process. We sought to resolve this question by comparing directly the levels of acetyl-CoA carboxylase and citrate cleavage enzyme in the rat mammary gland under conditions of the altered fatty acid synthesis accompanying lactation and its onset and cessation. The preparation of zoooo x g supernatant fractions from mammary extracts has been describedB. High-speed supernatant fractions were obtained from these by centrifuging at 105 ooo x g for I h and carefully withdrawing clear solution devoid of loosely sedimented particles or fat. Glycerol was added to the 20000 x g and 105ooo x supernatant solutions to 20% (v/v); these enzyme solutions are referred to as SpZO and SplO, respectively. Acetyl-CoA carboxylase was assayed as previously reportedB. Incorporation of H%O,was proportional to incubation time and enzyme concentration, at protein concentrations below 0.3 mg/mI. Experiments conducted with [Xlmalonyl-CoA under conditions of the acetyl-CoA carboxylase assay provided no evidence for the presence of an interfering malonyl-CoA decarboxylase, or a malonyl-CoA-CO, exchange reaction. Citrate cleavage enzyme was assayed spectrophotometrically by coupling with malate dehydrogenase ‘, (EC 1.1.1.37) which was present in great excess in all preparations. Kates of NADH oxidation were strictly proportional to protein concentration and linear with time. The milk content of each enzyme preparation was calculated from its lactose concentration6. Protein concentrations* of the enzyme solutions were corrected for milk protein by determining their lactose content, and relating this to the lactose and protein concentrations of rat milk Sp,, and Sp,,,,. Fig. I illustrates the similarity in the patterns of acetyl-CoA carboxylase and citrate cleavage enzyme activities throughout the lactation cycle, Roth enzymes rise rapidly during the first 4 days of lactation, remain at high levels and drop precipitously when the young are removed. This pattern, which parallels the alteration in fatty-acid synthesis in the mammary gland under the same conditions9suggests an important role for both enzymes in mammary fatty-acid synthesis. ,~BR.~HAM (cited in SREREIO), has demonstrated the concurrent alteration in the rate of fatty-acid synthesis and citrate cleavage enzyme activity during the lactation cycle in the rat mammary gland. Similar results were reported by LOWENSTEIN et aLlI. The specific activity of citrate cleavage enzyme is always higher than that of acetyl-Co.4 carB&him. Biophys.
Acta,
IOG (1965) 430-433